Isolated kidney cells and their use

Abstract

To provide isolated renal cells that include a renal tubule and a renal cell population producing erythropoietin (EPO), to provide methods for isolating and culturing the same, and to provide methods for treating a subject in need thereof with a cell population thereof.SOLUTION: Provided is an isolated human renal cell population for treating renal disease, the isolated human renal cell population including a higher proportion of tubular cells than that in human renal tissue or in cultured renal cells from human renal samples, and further including collecting duct cells, endocrine cells, vascular cells, and glomerular cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application was filed on November 12, 2008 under 35 U.S.C. § 119(e). No. 61 / 114,025, filed Nov. 12, 2008; No. 114,030, No. 61 / 201,056 filed December 5, 2008, No. 61 / 201,305 filed on December 8, 2008, and No. 61 / 201,305 filed on December 10, 2008 This application claims priority to US Pat. No. 61 / 121,311, filed on 2006-010144, which is hereby incorporated by reference. The subject matter of this application is filed on November 12, 2009. No. 61 / 260,833, filed on Oct. 13, 2003, the disclosure of which is incorporated herein by reference. Be absorbed.

[0002] Technical Field The present invention relates to a renal tubular cell population and a cell population that produces erythropoietin (EPO). US20130232633A1 - Isolated renal cells comprising a population of renal cells, and methods for isolating and culturing the same, as well as the cell population - Google Patents The present invention is directed to a method of treating a subject in need thereof using the same. [Background technology]

[0003] Background technology Chronic kidney disease (CKD) affects more than 19 million people in the United States and is associated with obesity, diabetes, and This is often the result of metabolic diseases involving diabetes and hypertension. Hypertension and insulin-independent diabetes, two diseases that are themselves on the rise around the world, It has been shown that this is caused by renal failure secondary to non-infectious diabetes mellitus (NIDDM). (United States Renal Data System:Costs o f CKD and ESRD. ed. Bethesda, MD, Nation al Institutes of Health, National Instit ute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238). Obesity, hypertension, and poor glycemic control have all been shown to be independent risk factors for kidney disease, causing glomerular and tubular lesions, proteinuria, and other systemic detectable changes in kidney filtration function (Aboushwareb, et al., World J Urol, 26: 295-300, 2008, Amann, K. et al., Nephrol Dial Transplant, 13: 1958-6 6, 1998). Patients with CKD in stages 1-3 of progression are managed by lifestyle changes and pharmacological interventions aimed at controlling underlying conditions (s), while patients in stages 4 -5 are managed by dialysis and usually a medication regimen that includes antihypertensive agents, erythropoiesis-stimulating agents (ESAs), iron and vitamin D supplementation. According to the United States Renal Data System ( USRDS), the average end-stage renal disease (ESRD) patient spends over $600 per month on injectable erythropoiesis-stimulating agents (ESAs), vitamin D supplements, and iron supplements (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, MD , National Institutes of Health, Nationa l Institute of Diabetes and Digestive an d Kidney Diseases, 2007 pp 223-238). Annual cost averaged over the years (65,405 US dollars), the medical cost for maintaining one patient exceeds 72,000 US dollars per year (United States Renal Da ta System: Costs of CKD and ESRD. ed. Be thesda, MD, National Institutes of Healt h, National Institute of Diabetes and Di gestive and Kidney Diseases, 2007 pp 223 -238). This figure only reflects the cost of standard treatment, and does not include treatment of other complications, emergency treatment, or auxiliary treatments such as the installation of vascular grafts for dialysis access. The total medical cost of CKD and ESRD in 2005 was 62 billion US dollars, accounting for 19% of the total medical cost of that year (United States Rena l Data System: Costs of CKD and ESRD. ed . Bethesda, MD, National Institutes of H . Bethesda, MD, National Institutes of Health, National Institute of Diabetes an d Digestive and Kidney Diseases, 2007 pp 223-238). For patients in stages 4-5, kidney transplantation is an effective option as a preventive measure to avoid dialysis or when dialysis is no longer sufficient to manage the condition. However, the number of stage 5 CKD patients (over 400,00 0) who can benefit from all kidney transplants in the United States is much larger than the number of suitable donor kidneys (about 16,000) available in any given year (United States Renal Data System: Costs of CKD and ESRD. ed. Bethesda, MD, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, 2007 pp 223-238). For patients in stages 4-5, kidney transplantation is an effective option as a preventive measure to avoid dialysis or when dialysis is no longer sufficient to manage the condition. However, the number of stage 5 CKD patients (over 400,000) who can benefit from all kidney transplants in the United States is much larger than the number of suitable donor kidneys (about 16,000) available in any given year far exceed (Powe, NR et al., Am J Kidney Dis, 53: S37-45,2009). Therefore, a new treatment paradigm is needed to delay or reduce dependence on dialysis and fill the gap caused by the shortage of donor kidneys.

[0004] Progressive kidney disease is caused by a combination of an initial disease (e.g., hypertension) and subsequent inappropriate renal responses to that disease. Such responses include the production of pro-inflammatory and profibrotic cytokines, as well as growth factors. Therefore, one strategy to delay the progression of CKD is to improve the inflammatory and fibrotic responses and reduce or reverse kidney degeneration by renal tissue repair and / or regeneration.

[0005] Chronic kidney disease affects not only humans but also some domestic animals. Patients suffering from kidney failure experience not only loss of kidney function (uremia) but also develop anemia due to a reduced ability of the bone marrow to produce a sufficient number of red blood cells (RBC) by erythropoiesis. Erythrocyte homeostasis depends on both the production of erythropoietin (EPO) by special interstitial fibroblasts present in the kidney and the ability of target erythroid progenitor cells in the bone marrow to produce more RBCs in response to EPO. Anemia in kidney failure is due to both a decrease in EPO production in the kidney and the adverse effects of uremic factors on the action of EPO in the bone marrow.

[0006] Previous clinical approaches to the treatment of chronic kidney disease have been dialysis and kidney transplantation for the recovery of kidney filtration and urine production, as well as recombinant EPO for the recovery of erythrocyte mass. or is accompanied by systemic delivery of EPO analogs. Dialysis improves survival rates for patients with mid- to late-stage kidney failure, but causes serious quality of life problems. Kidney transplantation is a highly desirable option (and often the only option) for patients with late-stage kidney failure, but the supply of high-quality donor kidneys does not meet the demand of the kidney failure population. Bolus administration of recombinant EPO to treat anemia is now associated with serious downstream health risks and has led to a black box warning from the FDA regarding this agent, necessitating further investigation of alternative treatments to restore erythrocyte homeostasis in this population. Preclinical studies have examined the in vivo efficacy and safety of cells producing EPO generated by gene therapy. These studies have shown that in vivo delivery of cells producing EPO can transiently stimulate erythropoiesis and RBC numbers. However, to date, none of these approaches have resulted in the regulation of erythrocyte homeostasis or long-term in vivo functionality. As a result, HCT and RBC numbers often increase above normal values, causing polycythemia vera and other complications. Delivery of cells producing EPO, which is therapeutically meaningful and offers advantages over the delivery of recombinant EPO, must not only increase HCT but also restore erythrocyte homeostasis without disrupting both positive and negative regulatory mechanisms. Anemia due to EPO deficiency is common in patients with kidney disease, but it is important to note that it can also occur as a result of other pathologies, including heart failure, multiple organ system failure, and other chronic diseases. for these patients, but causes serious quality of life problems. Kidney transplantation is a highly desirable option (and often the only option) for patients with late-stage kidney failure, but the supply of high-quality donor kidneys does not meet the demand of the kidney failure population. Treatment of anemia with recombinant EPO by bolus administration is now associated with serious downstream health risks and has led to a black box warning from the FDA regarding this agent, necessitating further investigation of alternative treatments to restore erythrocyte homeostasis in this population. Preclinical studies have examined the in vivo efficacy and safety of cells producing EPO generated by gene therapy. These studies have shown that in vivo delivery of cells producing EPO can transiently stimulate erythropoiesis and RBC numbers. However, to date, none of these approaches have resulted in the regulation of erythrocyte homeostasis or long-term in vivo functionality. As a result, HCT and RBC numbers often increase above normal values, causing polycythemia vera and other complications. Delivery of cells producing EPO, which is therapeutically meaningful and offers advantages over the delivery of recombinant EPO, must not only increase HCT but also restore erythrocyte homeostasis without disrupting both positive and negative regulatory mechanisms. Anemia due to EPO deficiency is common in patients with kidney disease, but it is important to note that it can also occur as a result of other pathologies, including heart failure, multiple organ system failure, and other chronic diseases. As a result, HCT and RBC numbers often increase above normal values, causing polycythemia vera and other complications. Delivery of cells producing EPO, which is therapeutically meaningful and offers advantages over the delivery of recombinant EPO, must not only increase HCT but also restore erythrocyte homeostasis without disrupting both positive and negative regulatory mechanisms. Anemia due to EPO deficiency is common in patients with kidney disease, but it is important to note that it can also occur as a result of other pathologies, including heart failure, multiple organ system failure, and other chronic diseases.

[0007] The kidney is a unique organ composed of many different (more than 10) specialized cell types​ All of these cell types originate developmentally from the intermediate mesoderm, but upon maturation, they form anatomical units that act in concert to provide morphologically and functionally distinct compartments, as well as regulation of endocrine functions such as blood filtration, urine production, acid-base regulation, and electrolyte balance, and production of erythropoietin (Epo), vitamin D, renin, and angiotensin. As demonstrated by the following examples, the intracellular compartments of the kidney are highly interdependent for homeostasis maintenance functions. The cells of the afferent arteriole act in concert with the specialized urinary tubule cells (macula densa) of the thick ascending limb of the loop of Henle to regulate blood flow through the glomerulus (Castrop, H. Acta Physiol (Oxf), 189:3-14, 2007). Protein handling by the kidney is coordinated by the fenestrated endothelial cells, podocytes, and basement membrane of the glomerulus, in conjunction with receptor-mediated endocytosis and protein reabsorption from glomerular filtrate by specialized proximal tubule cells (Jarad, G & Miner, JH. Curr Opin Nephrol Hypertens, 18:226-32, 2009). Production of active vitamin D by tubular cells regulates the homeostasis of interstitial cells through direct and indirect mechanisms that control extracellular matrix deposition, conversion of interstitial cells to myofibroblasts, and epithelial-mesenchymal transition (Tan, X, et al. J Steroid Biochem Mol Biol, 103:491-6, 2007). Despite their specific examples, all cell-cell interactions in the kidney are at least partly dependent on spatial and structural relationships. At the cellular level, the progression of CKD is due to cellular dysfunction or homeostasis and functionally distinct compartments, as well as regulation of endocrine functions such as blood filtration, urine production, acid-base regulation, and electrolyte balance, and production of erythropoietin (Epo), vitamin D, renin, and angiotensin. As demonstrated by the following examples, the intracellular compartments of the kidney are highly interdependent for homeostasis maintenance functions. The cells of the afferent arteriole act in concert with the specialized urinary tubule cells (macula densa) of the thick ascending limb of the loop of Henle to regulate blood flow through the glomerulus (Castrop, H. Acta Physiol (Oxf), 189:3-14, 2007). The kidney is highly interdependent for homeostasis maintenance functions. The cells of the afferent arteriole act in concert with the specialized urinary tubule cells (macula densa) of the thick ascending limb of the loop of Henle to regulate blood flow through the glomerulus (Castrop, H. Acta Physiol (Oxf), 189:3-14, 2007). The cells of the afferent arteriole act in concert with the specialized urinary tubule cells (macula densa) of the thick ascending limb of the loop of Henle to regulate blood flow through the glomerulus (Castrop, H. Acta Physiol (Oxf), 189:3-14, 2007). The kidney by the cells of the afferent arteriole acting in concert with the specialized urinary tubule cells (macula densa) of the thick ascending limb of the loop of Henle to regulate blood flow through the glomerulus (Castrop, H. Acta Physiol (Oxf), 189:3-14, 2007). Protein handling by the kidney is coordinated by the fenestrated endothelial cells, podocytes, and basement membrane of the glomerulus, in conjunction with receptor-mediated endocytosis and protein reabsorption from glomerular filtrate by specialized proximal tubule cells (Jarad, G & Miner, JH. C urr Opin Nephrol Hypertens, 18:226-32, 200 9). Protein handling by the kidney is coordinated by the fenestrated endothelial cells, podocytes, and basement membrane of the glomerulus, in conjunction with receptor-mediated endocytosis and protein reabsorption from glomerular filtrate by specialized proximal tubule cells (Jarad, G & Miner, JH. C urr Opin Nephrol Hypertens, 18:226-32, 200 9). Production of active vitamin D by tubular cells regulates the homeostasis of interstitial cells through direct and indirect mechanisms that control extracellular matrix deposition, conversion of interstitial cells to myofibroblasts, and epithelial-mesenchymal transition (Tan, X, et al. J Steroid Biochem Mol Biol, 103:491-6, 2007). Production of active vitamin D by tubular cells regulates the homeostasis of interstitial cells through direct and indirect mechanisms that control extracellular matrix deposition, conversion of interstitial cells to myofibroblasts, and epithelial-mesenchymal transition (Tan, X, et al. J Steroid Biochem Mol Biol, 103:491-6, 2007). Despite their specific examples, all cell-cell interactions in the kidney are at least partly dependent on spatial and structural relationships. At the cellular level, the progression of CKD is due to cellular dysfunction or homeostasis and structural relationships. At the cellular level, the progression of CKD is due to cellular dysfunction or homeostasis and structural relationships. At the cellular level, the progression of CKD is due to cellular dysfunction or homeostasis Loss of cell - cell interactions that maintain a specific cell type or loss of function of one or more cell types may occur. Thus, a good regenerative approach to the treatment of CKD would to some extent re - establish homeostasis through restoration of cell organization and cell - cell communication.

[0008] With the aim of reducing the morbidity and mortality associated with the progression of CKD, enhancement of specific renal functions such as tubular transport or Epo production is contemplated. Most of the cell - based treatment approaches for kidney diseases have focused on therapeutic interventions for acute renal failure (ARF) using stem cell types or progenitor cell types (Hopkins, C, et al. J Pathol,217: 265 - 81,2009). MSC (Humphreys BD & Bonventr e JV, Annu Rev Med 2008,59:311 - 325), endothelial progenitor cells (EPC) (Chade AR, et al., Circulation 20 09,119:547 - 557, Patschan D, et al.,Curr O pin Pharmacol 2006,6:176 - 183), and fetal cells or primordia of tissues (Hammerman MR, Curr Opin Nephrol Hy pertens 2001, 10:13 - 17, Kim SS, et al, St em Cells 2007, 25:1393 - 1401, Marshall D, et al., Exp Physiol 2007, 92:263 - 271, Yok oo T, et al., J Am Soc Nephrol 2006, 17: 1026 - 1034) have been delivered intrarenally immediately before or after ARF induction using various cell types including Numerous preclinical studies have been conducted, which may involve systemic delivery. For the treatment of ARF in humans, extracorporeal hollow fiber filters containing renal tubular cells have been tested as an adjunct to conventional dialysis (Ding, F & Humes, HD. Nephron Exp Nephrol, 109: e118 - 22, 2008, Humes, HD, et al. Kidney Int, 66: 1578 - 88, 2004, Humes, HD, et al. Nat Biotechnol, 17: 451 - 5, 1999). Transplantation of mesenchymal stem cells via the renal artery has also been clinically tested in patient populations at high risk of ARF episodes following cardiovascular surgical procedures (Westenfelder, C. Experimental Biology. New Orleans, LA, 2009). Limited preclinical studies have been conducted on cell - based therapeutic interventions for CKD (Chade, AR, et al. Circulation, 119: 547 - 57, 2009, Eliopoulos, N, et al.. J Am Soc Nephrol, 17: 1576 - 84, 2006, Kucic, T, et al.. Am J Physiol Renal Physiol, 295: F488 - 96, 2008). Combinations of fetal kidney anlagen + / - mesenchymal stem cells have been investigated in rodents (Yokoo, T, et al.. Transplantation, 85: 1654 - 8, 2008, Yokoo, T, et al. J Am Soc Nephrol, 17: 1026 - 34, 2006). Whole fetal kidney tissue transplanted into an appropriate environment such as the omentum, etc. develops into a kidney structure with limited function devices. devices have been tested (Ding, F & Humes, HD. Nephron E xp Nephrol, 109:e118 - 22, 2008, Humes, HD , et al. Kidney Int, 66: 1578 - 88, 2004, H umes, HD, et al. Nat Biotechnol, 17: 451 -5, 1999). Transplantation of mesenchymal stem cells via the renal artery has also been clinically tested in patient populations at high risk of ARF episodes following cardiovascular surgical procedures (W estenfelder, C. Experimental Biology. New Orleans, LA, 2009). Limited preclinical studies have been conducted on cell - based therapeutic interventions for CKD (Chade, AR, et al. Circula tion, 119: 547 - 57, 2009, Eliopoulos, N, et al. . J Am Soc Nephrol, 17: 1576 - 84, 2006, Kucic, T, et al.. Am J Physiol Renal Physiol, 295: . J Am Soc Nephrol, 17: 1576 - 84, 2006, Kucic, T, et al.. Am J Physiol Renal Physiol, 295: F488 - 96, 2008). Combinations of fetal kidney anlagen + / - mesenchymal stem cells have been investigated in rodents (Yokoo, T, et al.. Transpla ntation, 85: 1654 - 8, 2008, Yokoo, T, et al. ntation, 85: 1654 - 8, 2008, Yokoo, T, et al. J Am Soc Nephrol, 17: 1026 - 34, 2006) and whole fetal kidney tissue transplanted into an appropriate environment such as the omentum, etc. develops into a kidney structure with limited function It is clear that it can develop. However, as a component of the primordial fetal tissue the therapeutic role of MSC is unclear, and supplying human fetal kidney tissue for therapeutic purposes poses operational and ethical issues. In other studies, in a model of glomerulonephritis, protein loss , and Alport syndrome with fibrosis, irradiated COL4A3(- / - ) mice, when transplanted with cells derived from normal donor bone marrow, the progression in this model was partially delayed by replacing leaky glomerular podocytes with normal cells without mutations in the collagen gene (Prodromidi, EI, et al.. Stem Cells, 24: 2448-55, 2006, Sugimoto, H, e t al. Proc Natl Acad Sci U S A, 103: 732 1-6, 2006). Cell transplantation had a high evaluation for the stabilization of sCREAT, BUN, and sodium levels but no untreated / renal injury controls were presented for comparison in study 24. Chade et al. used a porcine model of unilateral renal artery stenosis to examine the effect of autologous EPC delivered into the kidney 6 weeks after injury (Chade AR, et al., Circulation 2009, 119:547-557). EPC somewhat improved tubulointerstitial fibrosis, significantly improved glomerulosclerosis, and improved renal blood flow but no change in blood pressure was observed with this treatment (Chade AR, et al., Circulation 2009, 119:547-557). So far studies examining the in vivo efficacy of cell-based CKD treatment have been transient and / or has produced partial effects but has gathered both systemic and histological evidence There is little research. Evidence of clinically significant benefits after intervention in the progression model of CKD Due to the limited number of studies providing doubts have been raised about the possibility of cell-based therapies fully restoring kidney function. However, regenerative therapies that stabilize kidney function and delay progression can address an unmet medical need within this patient population .

[0009] Reproducible in vivo model(s) of progressive CKD are essential for evaluating the therapeutic potential of candidate therapies. There are numerous models of ARF, including various tubular injuries induced chemically or by ischemia / reperfusion, but there are few models of progressive and end-stage CKD without significant intervention. The two-step 5 / 6 nephrectomy procedure in rats reproducibly generates end-stage and progressive states of renal insufficiency, accompanied by several important features of CKD, including hypertension, decreased glomerular filtration rate( GFR), increased serum creatinine (sCREAT) and BUN, glomerular interstitial and tubulointerstitial fibrosis, hyperlipidemia, hyperphosphatemia, and anemia, resulting in a systemic and histologically detectable disease(Kaufm an, JM, et al.. Kidney Int, 6: 10-7, 19742 2, Platt, R, et al. Clin Sci (Lond), 11: 2 17-31, 1952, Ormrod, D&Miller, T. Nephron, 26: 249-54, 1980, Brenner, BM. Am J Physiol, 249: F324-37, 1985). The presence of these clinically significant features demonstrates technical reproducibility 17-31, 1952, Ormrod, D&Miller, T. Nephron, 26: 249-54, 1980, Brenner, BM. Am J Physiol, 249: F324-37, 1985). The presence of these clinically significant features demonstrates technical reproducibility And in combination with commercial availability, provided a basis for selecting this model for the studies described herein.

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0010] Thus, there is a need for a new therapeutic paradigm that provides a substantial and durable enhancement of renal function to delay progression and improve quality of life in this patient population and reduce the annual cost burden on the healthcare system. Regenerative medicine technologies can provide alternative options for the next generation of treatment for CKD.

MEANS FOR SOLVING THE PROBLEMS

[0011] In one aspect, the present invention provides a mixture of human renal cells comprising a first cell population, B2, and a second cell population, wherein B2 comprises an isolated, enriched population of tubular cells, and the second cell population comprises cells that produce erythropoietin (EPO), glomerular cells, and vascular cells. In some embodiments, the mixture further comprises a third cell population. In one embodiment, the B2 cell population further comprises collecting duct epithelial cells. In one embodiment, the B2 cell population is hypoxia tolerant. In one embodiment, the B2 cell population can produce and / or stimulate the production of high molecular weight species of hyaluronic acid (HA) both in vitro and in vivo via the expression of HAS-2 (hyaluronic acid synthase-2). In another embodiment, the B2 cell population is iodixanol tolerant. In certain embodiments, the B2 cell population has a density of about 1.045 g / mL to about 1.052 g / mL. In a further embodiment, the second cell population is a B4 cell population. In one embodiment, B ​ The 4 - cell population has a density of from about 1.063 g / mL to about 1.091 g / mL. In other specific embodiments, the second cell population is a B3 cell population. In one embodiment, the B3 cell population has a density of from about 1.052 g / mL to about 1.063 g / mL. In still further embodiments, the mixture contains both B2 and B3 cell populations.

[0012] In still further embodiments, the mixture has had inactive or undesired components removed. In one embodiment, the mixture does not contain, or has had removed, a B1 cell population. In one embodiment, the B1 cell population is a collection of large granular cells of the collecting ducts and the renal tubule system having a density of less than about 1.045 g / mL. In other specific embodiments, the mixture does not contain, or has had removed, a B5 cell population. In one embodiment, the mixture does not contain a B5 cell population comprising low - granularity and low - viability debris and small cells having a density greater than about 1.091 g / mL. In certain embodiments, the mixture of cells provides stabilization and / or improvement and / or regeneration of renal function. In one embodiment, the mixture has the ability for receptor - mediated albumin uptake. In other embodiments, the mixture of cells has the ability for oxygen - regulated erythropoietin (EPO) expression. In still other embodiments, the mixture contains cells that express HAS - 2 and can produce, and / or stimulate the production of, high - molecular - weight species of hyaluronic acid (HA) both in vitro and in vivo. In one embodiment, the mixture can provide a regeneration stimulus upon delivery in vivo. In other embodiments, the mixture reduce, stabilize, or improve the decline of the reproductive and / or endocrine functions It can be done.

[0013] In all embodiments, the first and second cell populations can be derived from kidney tissue or cultured kidney cells. In one embodiment, B2 is megalin, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25 ), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (A qp1), aquaporin-2 (Aqp2), RAB17, member-RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), ion transport regulator 4 (Fxyd4) containing the FXYD domain, solute transporter family 9 (sodium / hydrogen exchanger), member-4 (Slc9a4), aldehyde dehydrogenase 3 family, member-B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member-A3 (Al dh1a3), and calpain-8 (Capn8). Another embodiment is characterized by the expression of the collecting duct marker aquaporin-4 (Aqp4). It is characterized by the expression of tubular cell markers selected from the group consisting of

[0014] In all embodiments, B4 can be characterized by the expression of vascular markers selected from the group consisting of PECAM, VEGF, KDR, and HIF1a. In other specific embodiments B4 can be characterized by the expression of the glomerular marker podocin (Podn) or nephrin (Neph). In another embodiment, B4 is unfractionated (UNFX), B ​​​​​​​​2. and being characterized by a population enriched in oxygen - regulatable EPO as compared to the B3 cell population This can be achieved. In another embodiment, B4 is chemokine (C - X - C motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), collagen, type V, alpha 2 (Col5a2), cadherin 5 (Cdh5), plasminogen activator , tissue (Plat), angiopoietin 2 (Angpt2), kinase insert domain protein receptor (Kdr), secreted protein, acidic, cysteine - rich (osteonectin) (Sparc), serglycin (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless - type MMTV integration site family member 4 (Wnt4), regulator of G - protein signaling 4 (Rgs4), platelet endothelial cell adhesion molecule (Pecam), and erythropoietin (Epo). It is characterized by the expression of markers selected from the group consisting of

[0015] In all embodiments, B3 is aquaporin 7 (Aqp7), ion transport regulator 2 (Fxyd2) containing an FXYD domain , solute carrier family 17 (sodium phosphate), member 3 (Slc17a3), solute carrier family 3, member 1 ( Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase (Napsa), solute carrier family 2 (facilitated glucose transporter), member 2 ( Slc2a2), alanyl (membrane) aminopeptidase (Anpep), transmembrane protein 2 7 (Tmem27), acyl - CoA synthetase medium - chain family member 2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose - 1,6 - bisphosphatase ​ - It can be characterized by the expression of a marker selected from the group consisting of ze1 (Fbp1) and alanine aminotransferase 2 (Agxt2). In other specific embodiments, B 3 is characterized by the vascular expression marker platelet endothelial cell adhesion molecule (Pecam) and the glomerular expression marker - podocin (Podn). - podocin (Podn).

[0016] In one aspect, the present invention provides a mixture of human kidney cells comprising a first cell population, B2, and a second cell population, wherein B2 comprises an isolated and enriched population of tubular cells, and the second cell population comprises one or more cell populations expressing the vascular expression marker platelet endothelial cell adhesion molecule (Pecam) and the glomerular expression marker - podocin (Podn). - podocin (Podn).

[0017] In another aspect, the present invention provides an isolated and enriched population of human kidney cells comprising a B2 cell population, wherein B2 comprises an isolated and enriched population of tubular cells. In one embodiment, B2 can produce and / or stimulate the production of high molecular weight species of hyaluronic acid (HA) both in vitro and in vivo through the expression of HAS-2 (hyaluronic acid synthase-2). In certain embodiments, the B2 cell population does not contain a B1 cell population comprising large granular cells of the collecting duct and tubule system having a density of less than about 1.04 5 g / mL. In another embodiment, the B2 cell population does not contain a B3 cell population comprising cells producing erythropoietin (EPO), glomerular cells, and vascular cells having a density of about 1.052 g / mL to about 1.0 63 g / mL. In yet another embodiment, the B2 cell population does not contain a B4 cell population having a density of about 1.063 g / mL to about 1.091 g / mL In yet another embodiment, the B2 cell population does not contain a B1 cell population comprising large granular cells of the collecting duct and tubule system having a density of less than about 1.04 5 g / mL. In another embodiment, the B2 cell population does not contain a B3 cell population comprising cells producing erythropoietin (EPO), glomerular cells, and vascular cells having a density of about 1.052 g / mL to about 1.0 63 g / mL. In yet another embodiment, the B2 cell population does not contain a B4 cell population having a density of about 1.063 g / mL to about 1.091 g / mL comprises a B4 cell population having a density of about 1.063 g / mL to about 1.091 g / mL It does not. In another embodiment, the B2 cell population does not include a B5 cell population containing low-granularity and low-viability debris and small cells having a density exceeding about 1.091 g / mL.

[0018] In another aspect, the present invention provides a method for preparing a human B2 cell population, comprising: a) exposing a cell suspension containing a non-concentrated heterogeneous kidney cell population to hypoxic culture conditions; and b) extracting a first cell fraction containing a B2 cell population. In one embodiment, the B2 cell population obtained by the method of the present invention contains a larger proportion of tubular cells and a smaller proportion of cells producing EPO, glomerular cells, and vascular cells, compared to the non-concentrated cell population. In another embodiment, the method further comprises contacting the cell suspension with a density gradient to separate one or more cell fractions between step a) and step b), and the first cell fraction is present in the gradient after centrifugation at a specific density between about 1.045 g / mL and about 1.052 g / mL.

[0019] In yet another aspect, the present invention provides a method for preparing a human B4 cell population, comprising: a) exposing a cell suspension containing a non-concentrated heterogeneous kidney cell population to hypoxic culture conditions; and b) extracting a first cell fraction containing a B4 cell population. In one embodiment, the B4 cell population obtained by the method of the present invention contains a larger proportion of cells producing EPO, vascular cells, and glomerular cells, and a smaller proportion of cells not producing EPO, non-vascular cells, and non-glomerular cells, compared to the non-concentrated cell population. In another embodiment, the method comprises separating one or more cell fractions between step a) and step b) to Further comprising the step of contacting the cell suspension with a density gradient, wherein the first cell fraction is present in the gradient after centrifugation at a specific density between about 1.063 g / mL and about 1.091 g / mL.

[0020] In another aspect, the present invention provides a method for preparing a human B3 cell population comprising: a) exposing a cell suspension comprising a non-concentrated heterogeneous kidney cell population to hypoxic culture conditions; and b) extracting a first cell fraction comprising a B3 cell population. In another embodiment, the method further comprises, between step a) and step b), the step of contacting the cell suspension with a density gradient to separate one or more cell fractions, wherein the first cell fraction is present in the gradient after centrifugation at a specific density between 1.052 g / mL and about 1.063 g / mL. Further comprising the step of contacting the cell suspension with a density gradient, wherein the first cell fraction is present in the gradient after centrifugation at a specific density between 1.052 g / mL and about 1.063 g / mL.

[0021] In another aspect, the present invention provides a method for generating a B2 cell preparation comprising: a) exposing a cell suspension comprising a non-concentrated heterogeneous kidney cell population to hypoxic culture conditions; b) applying the cell suspension to a flow cytometry instrument capable of simultaneously measuring forward scatter and side scatter in one or more individual cells within the cell population; c) selecting a subpopulation of cells from the cell population; d) sorting a subpopulation of cells from the cell population; and e) isolating a subpopulation of B4 cells from the cell population, wherein the subpopulation of B2 cells is characterized by high forward scatter and high side scatter compared to the majority of the population.

[0022] In another aspect, the present invention provides a method for generating a B2 cell preparation comprising: a) exposing a cell suspension comprising a non-concentrated heterogeneous kidney cell population to hypoxic culture conditions; b) applying the cell suspension to a flow cytometry instrument capable of simultaneously measuring forward scatter and side scatter in one or more individual cells within the cell population; Suitable for use in a flow cytometry instrument capable of simultaneously measuring forward scatter and side scatter in cells and c) selecting a subpopulation of cells from a cell population, d) sorting a subpopulation of cells from a cell population, and e) isolating a subpopulation of B4 cells from a cell population, to provide a method for generating a B4 cell preparation, wherein the subpopulation of B4 cells is characterized by low forward scatter and low side scatter compared to the majority of the population. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In yet another aspect, the present invention provides a transplantable construct for providing stable and / or improved renal function to a subject in need thereof, comprising a) a biomaterial comprising one or more biocompatible synthetic polymers or naturally occurring proteins or peptides, and b) a mixture of mammalian kidney cells comprising a first cell population, B2, and a second cell population, wherein the second cell population is coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial. In one embodiment, the second cell population coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial is B4. In another embodiment, the second cell population coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial is B3. In yet another embodiment, the second cell population coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial is characterized by low forward scatter and low side scatter compared to the majority of the population. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells.

[0023] In yet another aspect, the present invention provides a transplantable construct for providing stable and / or improved renal function to a subject in need thereof, comprising a) a biomaterial comprising one or more biocompatible synthetic polymers or naturally occurring proteins or peptides, and b) a mixture of mammalian kidney cells comprising a first cell population, B2, and a second cell population, wherein the second cell population is coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial. In one embodiment, the second cell population coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial is B4. In another embodiment, the second cell population coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial is B3. In yet another embodiment, the second cell population coated on, deposited on, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial is characterized by low forward scatter and low side scatter compared to the majority of the population. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. In all embodiments, forward scatter corresponds to the size of the cells. In all embodiments, side scatter corresponds to the granularity of the cells. The resulting, suspended therein, embedded therein, and / or otherwise combined therewith mixture further comprises a third cell population. In certain embodiments, the mixture coated on, deposited on or in, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial comprises B3 and B4. In all embodiments, the mixture may be derived from mammalian kidney tissue or cultured kidney cells. In one embodiment, the construct comprises a biomaterial configured as a three-dimensional (3-D) porous biomaterial suitable for capturing and / or adhering the mixture. In another embodiment, the construct comprises a biomaterial configured as a liquid or semi-fluid gel suitable for embedding, adhering, suspending, or coating mammalian cells. In yet another embodiment, the construct comprises a biomaterial comprising mainly high molecular weight species of hyaluronic acid (HA) in hydrogel form. In another embodiment, the construct comprises a biomaterial comprising mainly high molecular weight species of hyaluronic acid in the form of a porous foam. In yet another embodiment, the construct comprises a biomaterial comprising HA molecules in the size range of 5.1 kDa to greater than 2 x 106 kDa. In yet another embodiment, the construct comprises a biomaterial comprising a polylactic acid-based foam having pores of about 50 microns to about 300 microns. In another embodiment, the construct comprises one or more cell populations derived from an autologous kidney sample. In one embodiment, the kidney sample is a kidney biopsy. In a further embodiment, the construct comprises one or more cell populations derived from a non-autologous kidney sample. In one embodiment, the construct provides erythrocyte homeostasis. The resulting, suspended therein, embedded therein, and / or otherwise combined therewith mixture further comprises a third cell population. In certain embodiments, the mixture coated on, deposited on or in, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial comprises B3 and B4. The resulting, suspended therein, embedded therein, and / or otherwise combined therewith mixture further comprises a third cell population. In certain embodiments, the mixture coated on, deposited on or in, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial comprises B3 and B4. The resulting, suspended therein, embedded therein, and / or otherwise combined therewith mixture further comprises a third cell population. In certain embodiments, the mixture coated on, deposited on or in, captured in, suspended in, embedded in, and / or otherwise combined with the biomaterial comprises B3 and B4. In all embodiments, the mixture may be derived from mammalian kidney tissue or cultured kidney cells. In one embodiment, the construct comprises a biomaterial configured as a three-dimensional (3-D) porous biomaterial suitable for capturing and / or adhering the mixture. In all embodiments, the mixture may be derived from mammalian kidney tissue or cultured kidney cells. In one embodiment, the construct comprises a biomaterial configured as a three-dimensional (3-D) porous biomaterial suitable for capturing and / or adhering the mixture. In another embodiment, the construct comprises a biomaterial configured as a liquid or semi-fluid gel suitable for embedding, adhering, suspending, or coating mammalian cells. In yet another embodiment, the construct comprises a biomaterial comprising mainly high molecular weight species of hyaluronic acid (HA) in hydrogel form. In another embodiment, the construct comprises a biomaterial configured as a liquid or semi-fluid gel suitable for embedding, adhering, suspending, or coating mammalian cells. In yet another embodiment, the construct comprises a biomaterial comprising mainly high molecular weight species of hyaluronic acid (HA) in hydrogel form. In another embodiment, the construct comprises a biomaterial configured as a liquid or semi-fluid gel suitable for embedding, adhering, suspending, or coating mammalian cells. In yet another embodiment, the construct comprises a biomaterial comprising mainly high molecular weight species of hyaluronic acid (HA) in hydrogel form. In another embodiment, the construct comprises a biomaterial comprising mainly high molecular weight species of hyaluronic acid (HA) in the form of a porous foam. In yet another embodiment, the construct comprises a biomaterial comprising HA molecules in the size range of 5.1 kDa to greater than 2 x 106 kDa. In another embodiment, the construct comprises a biomaterial comprising mainly high molecular weight species of hyaluronic acid (HA) in the form of a porous foam. In yet another embodiment, the construct comprises a biomaterial comprising HA molecules in the size range of 5.1 kDa to greater than 2 x 106 kDa. In yet another embodiment, the construct comprises a biomaterial comprising HA molecules in the size range of 5.1 kDa to greater than 2 x 106 kDa. In yet another embodiment, the construct comprises a biomaterial comprising a polylactic acid-based foam having pores of about 50 microns to about 300 microns. In yet another embodiment, the construct comprises a biomaterial comprising HA molecules in the size range of 5.1 kDa to greater than 2 x 106 kDa. In yet another embodiment, the construct comprises a biomaterial comprising a polylactic acid-based foam having pores of about 50 microns to about 300 microns. In yet another embodiment, the construct comprises a biomaterial comprising a polylactic acid-based foam having pores of about 50 microns to about 300 microns. In another embodiment, the construct comprises one or more cell populations derived from an autologous kidney sample. In yet another embodiment, the construct comprises a biomaterial comprising a polylactic acid-based foam having pores of about 50 microns to about 300 microns. In another embodiment, the construct comprises one or more cell populations derived from an autologous kidney sample. In another embodiment, the construct comprises one or more cell populations derived from an autologous kidney sample. In one embodiment, the kidney sample is a kidney biopsy. In another embodiment, the construct comprises one or more cell populations derived from an autologous kidney sample. In one embodiment, the kidney sample is a kidney biopsy. In a further embodiment, the construct comprises one or more cell populations derived from a non-autologous kidney sample. In one embodiment, the construct provides erythrocyte homeostasis.

[0024] In yet another aspect, the present invention provides a method for treating kidney disease in a subject in need thereof. a) a mixture of mammalian kidney cells comprising a first cell population, B2, and a second cell population; administering to the subject a composition comprising the compound comprising: and determining in a test sample from the subject that the difference in the indicator level differs from the difference in the indicator level. indicates a reduction, stabilization, or amelioration of decline in one or more renal functions in a subject. In one embodiment, the method includes a mixture of cells comprising a third cell population. In another embodiment, the second cell population is B4 or B3. In certain embodiments, the kidney disease treated by the methods of the present invention is erythrocyte sequestration. In certain embodiments, the EPO deficiency is anemia. In some embodiments, the EPO deficiency or anemia is secondary to renal failure in a subject. In some other embodiments, the EPO deficiency or anemia is caused by chronic renal failure, primary chronic EPO deficiency, chemotherapy or antiviral treatment, non-myeloid cancer, HIV infection, liver disease , heart failure, rheumatoid arthritis, or multiple organ system failure. In certain embodiments, the composition used in the method comprises one or more biocompatible Biomaterials including synthetic polymers and / or naturally occurring proteins or peptides are also included. The mixture may be coated on, deposited on or within a biomaterial or may be entrapped therein. may be suspended therein, embedded therein, and / or otherwise combined therewith. do.

[0025] In yet another aspect, the present invention relates to a cell preparation and a mixture thereof, or to a subject in need thereof. The present invention provides the use of an implantable structure for the preparation of a drug useful for the treatment of kidney diseases, anemia, or EPO deficiency in an elephant. Provide the use of an implantable structure.

[0026] In one aspect, the present invention provides a selected population of renal cells that can be separated by density gradient centrifugation using a gradient containing a fraction having a density of about 1.045 g / mL to about 1.052 g / mL after being exposed to an oxygen level of about 1% to about 5% for about 12 to about 24 hours. The cell population is (i) retained in the gradient after centrifugation at a density of 1.045 g / mL to about 1.052 g / mL, (ii) contains a cell population of the renal tubules of the kidney characterized by the expression of at least one renal tubule cell marker, (iii) contains a subpopulation of renal tubule cells of the kidney capable of receptor-mediated albumin transport, and (iv) can regulate one or more renal functions when delivered to a subject at risk of or having kidney disease. After being exposed to an oxygen level of about 1% to about 5% for about 12 to about 24 hours, the cell population is separated by density gradient centrifugation using a gradient containing a fraction having a density of about 1.045 g / mL to about 1.052 g / mL. Provide a selected population of renal cells. The cell population is retained in the gradient after centrifugation at a density of 1.045 g / mL to about 1.052 g / mL. The cell population contains a cell population of the renal tubules of the kidney characterized by the expression of at least one renal tubule cell marker. The cell population contains a subpopulation of renal tubule cells of the kidney capable of receptor-mediated albumin transport. When delivered to a subject at risk of or having kidney disease, the cell population can regulate one or more renal functions. When delivered to a subject at risk of or having kidney disease, the cell population can regulate one or more renal functions.

[0027] In yet another aspect, the present invention provides a selected population of renal cells that can be separated by density gradient centrifugation using a gradient containing a fraction having a density of about 1.063 g / mL to about 1.091 g / mL after being exposed to an oxygen level of about 1% to about 5% for about 12 hours to about 24 hours. The cell population is (i) retained in the gradient after centrifugation at a density of 1.063 g / mL to about 1.091 g / mL, (ii) contains cells expressing oxygen-regulated erythropoietin (EPO), glomerular cells, and vascular cells, (iii) can regulate one or more renal functions when delivered to a subject at risk of or having kidney disease, and (iv) can enhance the regulation of one or more renal functions by the population of renal cells according to claim 65 when administered simultaneously. After being exposed to an oxygen level of about 1% to about 5% for about 12 hours to about 24 hours, the cell population is separated by density gradient centrifugation using a gradient containing a fraction having a density of about 1.063 g / mL to about 1.091 g / mL. Provide a selected population of renal cells. The cell population is retained in the gradient after centrifugation at a density of 1.063 g / mL to about 1.091 g / mL. The cell population contains cells expressing oxygen-regulated erythropoietin (EPO), glomerular cells, and vascular cells. When delivered to a subject at risk of or having kidney disease, the cell population can regulate one or more renal functions. When delivered to a subject at risk of or having kidney disease, the cell population can regulate one or more renal functions. When administered simultaneously, the cell population can enhance the regulation of one or more renal functions by the population of renal cells according to claim 65. When administered simultaneously, the cell population can enhance the regulation of one or more renal functions by the population of renal cells according to claim 65.

[0028] In yet another aspect, the present invention provides a population of renal cells, the cells being:i) placed in adherent culture on a standard tissue-culture-treated plastic dish at an initial density of 25,000 cells / cm 2 in a medium consisting of a 1:1 mixture of high-glucose DMEM and fully supplemented KSFM with 5% fetal bovine serum at 37 °C and 21% oxygen for a period of between 24 and 72 hours; ii) exposed to a 50 - 100% medium change with the same medium and cultured at 37 °C and 2% oxygen for 18 to 24 hours; iii) collected via trypsinization, resuspended and washed with serum-free KSFM medium or PBS; iv) loaded onto a prepared density gradient, the gradient containing layers of defined density from 1.045 g / mL to about 1.052 g / mL, at least one layer of greater density and at least one layer of lesser density, the gradient being prepared in a 15 mL conical tube with a total volume of at least 5, at most 14 mL of liquid, and the number of cells loaded onto the gradient being at least 50 million but not exceeding 100 million, v ) the gradient is subjected to centrifugation at 800 x G for 20 - 30 minutes without braking; separated at a density of 1.045 g / mL to 1.052 g / mL; and / or megalin, cubilin, hyaluronan synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP 2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin -1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member-RAS oncogene family- (Rab17), GATA binding protein 3 (Gata3), FXYD domain containing ion transport regulator-4 (Fxyd4), solute carrier family-9 (sodium -dependent glucose transporter), member 5 (Slc9a5), solute carrier family-22 (organic anion transporter family), member 17 (Slc22a17), and / or solute carrier family-22 (organic anion transporter family), member 20 (Slc22a20); -1 (Aqp1), aquaporin-2 (Aqp2), RAB17, member-RAS oncogene family- (Rab17), GATA binding protein 3 (Gata3), FXYD domain containing ion transport regulator-4 (Fxyd4), solute carrier family-9 (sodium lithium / hydrogen exchanger), member-4 (Slc9a4), aldehyde dehydrogenase 3 family -, member-B1 (Aldh3b1), aldehyde dehydrogenase 1 family-, member-A 3 (Aldh1a3), calpain-8 (Capn8), and aquaporin-4 (Aq p4) marker selected from the group consisting of; and / or kidney disease stabilize, reduce its decline, or improve one or more renal functions of an immunocompatible subject suffering from

[0029] In another aspect, the present invention provides a population of renal cells, the cells being i) 24 to 72 hours at 37 °C and 21% oxygen for a period of 5% fetal bovine serum, high glucose DMEM and placed in adherent culture on a standard tissue-culture-treated plastic dish at an initial density of 25,000 cells / cm 2 in a medium consisting of a 1:1 mixture of fully supplemented KSFM; ii) exposed to a 50-100% medium change with the same medium and cultured at 37 °C and 2% oxygen for 18 to 24 hours; iii) collected via trypsinization, resuspended, and washed with serum-free KSFM medium or PBS; iv) loaded onto a prepared density gradient, the gradient containing layers of defined density from 1.063 g / mL to about 1.091 g / mL, larger at least one layer of density and at least one layer of lower density, whereby the gradient is prepared in a 15 mL conical tube with a total volume of at least 5 and at most 14 mL of liquid, and the number of cells loaded onto the gradient is at least 50 million but does not exceed 100 million; v) the gradient is disrupted by centrifugation at 800 x G for 20 to 30 minutes without braking; 1.063 g / mL to about 1.091 g / mL density; and / or VEGF, KDR, HIF1a, Podocin (Podn) or Nephrin (Neph), chemokine (C-X -C motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), collagen, type V, alpha 2 (Col5a2), cadherin 5 (Cdh5), plas minogen activator, tissue (Plat), angiopoietin 2 (Angpt2), chi nase insert domain protein receptor (Kdr), secreted protein, acidic, cyste ine-rich (osteonectin) (Sparc), serglycin (Srgn), TIMP metallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless type MMTV integration site family, member 4 (Wnt4), G-protein signaling 4 (Rgs4) regulator, platelet endothelial cell adhesion molecule (Pecam), and erythropoietin (Epo) characterized by a marker selected from the group consisting of; and / or one or more renal functions of an immunocompatible subject suffering from a kidney disease can be stabilized, its decline reduced, or improved.

[0030] In one aspect, the present invention provides an isolated population of kidney cells that produce erythropoietin (EPO). In one embodiment, the population is an isolated, enriched population of mammalian cells that produce EPO. In another embodiment, the population is an isolated, enriched population of mammalian cells that produce erythropoietin (EPO) and contains a greater proportion of cells that produce EPO than an unenriched population of mammalian cells that produce EPO . The population can be derived from kidney tissue or cultured kidney cells. The population can be obtained from the kidney of a subject

[0031] of a subject. It can be derived from a sample. The sample can be kidney tissue or cultured kidney cells derived from a kidney sample obtained from a subject. In another embodiment, the cell population contains a greater proportion of cells that produce EPO than an unenriched population of mammalian cells that produce EPO. In yet another embodiment, the cell population contains a smaller proportion of kidney tubule cells than an unenriched population of mammalian cells that produce erythropoietin (EPO). In all embodiments, the cell population can be enriched for cells that produce EPO. In all embodiments, the cell population can be depleted of cells that do not produce EPO. In all embodiments, the cell population can be enriched for kidney tubule cells. In another aspect, the present invention provides a cell population of cells that produce erythropoietin (EPO) that biologically respond under specific culture conditions. In yet another embodiment, the biological responsiveness is the induction of EPO expression when the cell population is cultured under hypoxic conditions compared to a cell population cultured under non-hypoxic conditions. In yet another embodiment, the biological responsiveness is an increase in EPO expression when the cell population is cultured under hypoxic conditions compared to a cell population cultured under non-hypoxic conditions. In some embodiments, the hypoxic culture conditions include, but are not limited to, reducing the available oxygen level in the culture system compared to a cell population cultured under conditions where the oxygen level is not reduced for the cell population. In yet another embodiment, the reduction in the available oxygen level is less than about 5% and the oxygen level is not reduced.

[0032] In all embodiments, the cell population can be enriched for cells that produce EPO. In all embodiments, the cell population can be depleted of cells that do not produce EPO. In all embodiments, the cell population can be enriched for kidney tubule cells.

[0033] In another aspect, the present invention provides a cell population of cells that produce erythropoietin (EPO) that biologically respond under specific culture conditions. In yet another embodiment, the biological responsiveness is the induction of EPO expression when the cell population is cultured under hypoxic conditions compared to a cell population cultured under non-hypoxic conditions. In yet another embodiment, the biological responsiveness is an increase in EPO expression when the cell population is cultured under hypoxic conditions compared to a cell population cultured under non-hypoxic conditions. In some embodiments, the hypoxic culture conditions include, but are not limited to, reducing the available oxygen level in the culture system compared to a cell population cultured under conditions where the oxygen level is not reduced for the cell population. In yet another embodiment, the biological responsiveness is the induction of EPO expression when the cell population is cultured under hypoxic conditions compared to a cell population cultured under non-hypoxic conditions. In yet another embodiment, the biological responsiveness is an increase in EPO expression when the cell population is cultured under hypoxic conditions compared to a cell population cultured under non-hypoxic conditions. In some embodiments, the hypoxic culture conditions include, but are not limited to, reducing the available oxygen level in the culture system compared to a cell population cultured under conditions where the oxygen level is not reduced for the cell population. In yet another embodiment, the reduction in the available oxygen level is less than about 5% and the oxygen level is not reduced. In yet another embodiment, the reduction in the available oxygen level is less than about 5% and the oxygen level is not reduced. ​The condition is the oxygen level in the atmosphere (about 21%). In another embodiment, the increase in EPO expression is observed at an oxygen level lower than that in the atmosphere (21%) when compared to cultures tested at levels of 21% or higher. In another embodiment, the level of induction and / or increased expression of EPO is observed when culturing cells under hypoxic culture conditions of about less than 5% oxygen, i.e., compared to cells cultured under atmospheric oxygen levels (about 21%), i.e., non-hypoxic culture conditions. In one embodiment, the EPO expression that responds biologically to hypoxic conditions is controlled by the hypoxia inducible factor HIF. In another embodiment, the EPO expression that responds biologically to hypoxic conditions is controlled by HIF1α. In yet another embodiment, the EPO expression that responds biologically to hypoxic conditions is controlled by the hypoxia inducible factor HIF2α.

[0034] In one embodiment, the biological responsiveness is the induction of EPO expression when culturing the cell population via perfusion, compared to a cell population not cultured via perfusion. In another embodiment, the biological responsiveness is the increase in the expression of EPO when compared to a cell population not cultured via perfusion. In some embodiments, the perfusion conditions include, but are not limited to, transient, intermittent, or continuous circulation or agitation of the liquid to transmit power to the cells via the flow of the liquid. In another embodiment, the perfusion culture conditions are maintained such that the cell population is cultured in or on a material that provides a framework and / or space that allows for the formation of a three-dimensional structure.

[0035] In another aspect, the present invention provides a kidney cell containing the cell population described herein. Provide a mixture or combination of cells. In one embodiment, the cell mixture is enriched for a first cell population that produces EPO and not enriched for cells that produce EPO Contains a second cell population. In certain other embodiments, the second cell population can contain one or more of the following One or more types of kidney-derived cells that can contain one or more of: cells derived from the renal tubules, cells derived from the glomeruli, cells derived from the interstitium, cells derived from the collecting ducts, cells derived from connective tissue, cells derived from blood, or cells derived from blood vessels. In another embodiment, the second cell population is enriched for kidney tubule cells.

[0036] In all embodiments, the kidney tubule cells described herein can include, but are not limited to, one or more of the following, characterized by the expression of tubule cell markers: hyaluronan synthase 2 (HAS2), CYP2D25 (vitamin D3 25-hydroxylase), megalin, cubilin, N-cadherin, E-cad herin, aquaporin-1, aquaporin-2, RAB17, member-RAS oncogene family amil (Rab17), GATA binding protein 3 (Gata3), ion transport regulator 4 (Fxyd4) containing the FXYD domain, solute transporter family 9 (sodium / hydrogen exchanger), member-4 (Slc9a4), aldehyde dehydrogenase 3 family, member-B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member-A3 ( Aldh1a3), and calpain-8 (Capn8).

[0037] In one aspect, the present invention provides an isolated, enriched population of mammalian kidney tubule cells ​​​​​​​For use. In one embodiment, the isolated cell population is enriched for mammalian cells that produce at least some EPO and contain renal tubular cells of the kidney. In another embodiment, the cell population has a higher proportion of tubular cells than an unenriched population containing tubular cells. In certain other embodiments, the isolated, enriched population of mammalian kidney tubular cells contains a higher proportion of tubular cells than an unenriched population containing tubular cells and mammalian cells that produce at least some EPO. In another embodiment, the enriched population of mammalian kidney tubular cells has a relatively low proportion of cells that produce EPO removed compared to an unfractionated, heterogeneous mixture or a population enriched for cells that produce EPO. In all embodiments, the type of source from which the kidney cell population or mixture of kidney cell populations described herein is derived can be autologous or allogeneic, syngeneic (autologous or syngeneic transplantation), and any combination thereof. For example, in some embodiments, the cell mixture can contain (i) a first cell population derived from an autologous source and a second cell population derived from an autologous source, or (ii) a first cell population derived from an autologous source and a second cell population derived from an allogeneic source. In another aspect, the present invention provides a method for generating a cell population enriched for cells that produce EPO. In one embodiment, the method comprises: a) preparing a cell suspension having an unenriched, heterogeneous population of rodent kidney cells from mechanically dissociated or enzymatically digested mammalian kidney tissue; b) separating one or more cell fractions based on buoyant density;

[0038]

[0039] ​​​​​​​​​​​​​​​​ contacting the cell suspension with a density gradient; c) centrifuging the cell suspension of step b) to define one or more cell fractions; and d) extracting a first cell fraction containing a concentrated cell population, wherein the concentrated cell population produces a higher proportion of cells producing EPO and a lower proportion of cells not producing EPO compared to the non-concentrated cell population. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.025 g / mL and about 1.035 g / mL, or less than about 1.045 g / mL. In another embodiment, the first cell fraction of step d) is present in the gradient after centrifugation at a specific density between about 1.025 g / mL and about 1.035 g / mL, or less than about 1.045 g / mL. In certain other embodiments, the centrifugation of step (c) further produces at least one additional cell fraction that is not concentrated relative to the concentrated cell population that produces EPO. In another embodiment, the method further includes step e) of extracting at least one additional cell fraction. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.062 g / mL and about 1.088 g / mL. In another embodiment, the additional cell fraction is present in the gradient after centrifugation at a specific density between about 1.062 g / mL and about 1.088 g / mL. In another embodiment, the method comprises: a) preparing a cell suspension from a cultured non-concentrated heterogeneous mammalian cell population comprising at least some cells that express or are capable of expressing EPO; b) contacting the cell suspension with a density gradient to separate one or more cell fractions based on buoyant density; c) centrifuging the cell suspension of step b) to define one or more cell fractions; and d) extracting a first cell fraction containing a concentrated cell population, wherein the concentrated cell population produces a higher proportion of cells producing EPO and a lower proportion of cells not producing EPO compared to the non-concentrated cell population. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.025 g / mL and about 1.035 g / mL, or less than about 1.045 g / mL. In another embodiment, the first cell fraction of step d) is present in the gradient after centrifugation at a specific density between about 1.025 g / mL and about 1.035 g / mL, or less than about 1.045 g / mL. In certain other embodiments, the centrifugation of step (c) further produces at least one additional cell fraction that is not concentrated relative to the concentrated cell population that produces EPO. In another embodiment, the method further includes step e) of extracting at least one additional cell fraction. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.062 g / mL and about 1.088 g / mL. In another embodiment, the additional cell fraction is present in the gradient after centrifugation at a specific density between about 1.062 g / mL and about 1.088 g / mL. In another embodiment, the method further includes step e) of extracting at least one additional cell fraction. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.062 g / mL and about 1.088 g / mL. In another embodiment, the additional cell fraction is present in the gradient after centrifugation at a specific density between about 1.062 g / mL and about 1.088 g / mL. In another embodiment, the method comprises: a) preparing a cell suspension from a cultured non-concentrated heterogeneous mammalian cell population comprising at least some cells that express or are capable of expressing EPO; b) contacting the cell suspension with a density gradient to separate one or more cell fractions based on buoyant density;

[0040] In another embodiment, the method comprises: a) preparing a cell suspension from a cultured non-concentrated heterogeneous mammalian cell population comprising at least some cells that express or are capable of expressing EPO; b) contacting the cell suspension with a density gradient to separate one or more cell fractions based on buoyant density; c) centrifuging the cell suspension of step b) to define one or more cell fractions; and d) extracting a first cell fraction containing a concentrated cell population, wherein the concentrated cell population produces a higher proportion of cells producing EPO and a lower proportion of cells not producing EPO compared to the non-concentrated cell population. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.025 g / mL and about 1.035 g / mL, or less than about 1.045 g / mL. contacting the pairs, c) centrifuging the cell suspension of step b) to define one or more cell fractions, and d) extracting a first cell fraction containing the concentrated cell population, wherein the concentrated cell population has a greater proportion of cells producing EPO and a smaller proportion of cells not producing EPO compared to the non-concentrated cell population. In one embodiment, the cell suspension of step a) is obtained from a population of cultured non-concentrated heterogeneous murine cells. In such an embodiment, the density gradient includes a layer of a specific density between about 1.073 g / mL and about 1.091 g / mL. In one embodiment, the first cell fraction of step d) is present in the gradient after centrifugation at a specific density between about 1.073 g / mL and about 1.091 g / mL. In certain other embodiments, the centrifugation of step (c) further produces at least one additional cell fraction that is not concentrated relative to the concentrated cell population producing EPO. In another embodiment, the method further includes step e) of extracting at least one additional cell fraction. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.041 g / mL and about 1.062 g / mL. In another embodiment, the additional cell fraction is present in the gradient after centrifugation at a specific density between about 1.041 g / mL and about 1.062 g / mL. centrifuging, and d) extracting a step of a first cell fraction containing the concentrated cell population, wherein the concentrated cell population has a larger proportion when compared with the non-concentrated cell population, of cells producing EPO and a smaller proportion of cells not producing EPO. In one embodiment, the cell suspension of step a) is a non-cultured, concentrated heterogeneous rodent cell population. In such an embodiment, the density gradient is about 1.073 g / mL to about 1.091 g / mL including a layer of a specific density. In one embodiment, the first cell fraction of step d) is about 1.073 g / mL to about 1.091 g / mL of a specific density after centrifugation and is present in the gradient. In certain other embodiments, the centrifugation of step (c) produces at least one additional cell fraction that is not concentrated relative to the concentrated cell population producing EPO. In another embodiment, the method further includes step e) of extracting at least one additional cell fraction. In certain other embodiments, the density gradient includes a layer of a specific density between about 1.041 g / mL and about 1.062 g / mL. In another embodiment, the additional cell fraction is about 1.041 g / mL to about 1.062 g / mL of a specific density after centrifugation and is present in the gradient. In some embodiments, the density gradient includes a layer of a specific density between about 1.041 g / mL and about 1.062 g / mL. In another embodiment, the additional cell fraction is about 1.041 g / mL to about 1.062 g / mL of a specific density after centrifugation and is present in the gradient.

[0041] In some embodiments, the concentrated cell population is concentrated with respect to cells producing EPO and cells not producing EPO are removed. In other embodiments, the concentrated cell population is concentrated with respect to interstitial fibroblasts and tubular cells and collecting duct cells are removed. In another embodiment, the concentrated cell population is cells producing EPO, glomerular cells, ​​​ and concentrated for vascular cells. In another embodiment, the centrifugation of step (c) generates at least one additional cell fraction that is not concentrated for the concentrated cell population. In some embodiments, the additional cell fraction, when compared to the non-concentrated cell population, contains a larger proportion of cells that do not produce EPO and a smaller proportion of cells that produce EPO. In one embodiment, the at least one additional cell fraction has a smaller proportion of cells that produce EPO compared to the first cell fraction. In another embodiment, the additional cell fraction contains a larger proportion of renal tubular cells of the kidney compared to the first cell fraction.

[0042] In some embodiments, the density gradient used to generate a cell population concentrated for cells that produce EPO is a density gradient of iodixanol.

[0043] In another aspect, the present invention provides a method for generating a concentrated population of cells that produce erythropoietin E PO using flow cytometry. In one embodiment, the method comprises: a) preparing a cell suspension containing a non-concentrated heterogeneous population of kidney cells from mechanically dissociated or enzymatically digested mammalian kidney tissue; b) applying the cell suspension to a flow cytometry instrument capable of simultaneously measuring forward scatter and side scatter of one or more individual cells within the cell population; c) selecting a cell subpopulation from the cell population; d) sorting a cell subpopulation from the cell population; and e) isolating a cell subpopulation from the cell population, the cell subpopulation being characterized by low forward scatter and low side scatter compared to the whole population.

[0044] In another embodiment, the method comprises: a) preparing a cell suspension from a population of cultured mammalian cells comprising at least some cells that express or are capable of expressing EPO; b) providing the cell suspension to a flow cytometry instrument capable of simultaneously measuring forward scatter and side scatter in one or more individual cells within the cell population; c) selecting a cell subpopulation from the cell population; d) sorting a cell subpopulation from the cell population; e) isolating a cell subpopulation from the cell population, wherein the cell subpopulation is characterized by low forward scatter and low side scatter compared to the whole population.

[0045] In yet another embodiment, the forward scatter corresponds to the size of the cell. In one embodiment, the side scatter corresponds to the granularity of the cell. Other embodiments of the invention provide a concentrated cell fraction that is: i) enriched for cells that produce EPO and depleted of cells that do not produce EPO, or ii) enriched for specific interstitial cortical fibroblasts that produce EPO and depleted of epithelial cells. In certain other embodiments, the selection step c) comprises generating at least one additional fraction that is not enriched for the cell population. In another embodiment, the additional fraction contains a smaller proportion of cells that produce EPO compared to the fraction enriched for EPO. In a further aspect of the invention, the method further comprises a step of culturing in vitro. In one embodiment, the concentrated cell population is cultured in vitro after isolation. In another embodiment, the culturing step is on a two-dimensional surface of glass or plastic suitable for culturing mammalian cells in a medium adapted to support the growth and / or maintenance of the cell population.

[0046] Including the use of monolayer culture. In other embodiments, the culturing step comprises maintaining and / or culturing the cells on a three-dimensional (3D) scaffold suitable for growth. In certain other embodiments the scaffold contains one or more biocompatible synthetic polymers or naturally occurring proteins or peptides. In another embodiment, the scaffold is configured as a porous scaffold suitable for capturing or adhering mammalian cells. In other embodiments, the scaffold is configured as a gel suitable for embedding, adhering to, or coating mammalian cells.

[0047] In another aspect, the invention provides a method comprising culturing under perfusion conditions. In one embodiment, the perfusion conditions include, but are not limited to, transient, intermittent or continuous circulation or agitation of a liquid to convey power to the cells via the liquid flow. In another embodiment the perfusion culture conditions are maintained such that the cell population is cultured in or on a material that provides a framework and / or space that allows the formation of a three-dimensional structure.

[0048] In another aspect, the invention provides a method comprising culturing under hypoxic conditions. In some embodiments, the hypoxic culture conditions include, but are not limited to, reducing the available oxygen level in the culture system compared to a cell population cultured under conditions where the oxygen level is not reduced for the cell population. In certain other embodiments, the reduction in the available oxygen level is less than about 5% and the condition where the oxygen level is not reduced is the oxygen level in the atmosphere (about 21%). In another embodiment, the reduced oxygen condition is represented by an oxygen level of less than 21% (in the atmosphere).

[0049] In another additional aspect, the present invention provides a method comprising the step of measuring EPO expression in a cell population. In all embodiments, the EPO expression is EPO mRNA expression. In all embodiments, the EPO expression is detectable and / or is detected. In another embodiment, the EPO expression is induced in a cell population cultured via perfusion as compared to a cell population not cultured via perfusion. In other embodiments, the EPO expression is induced in a cell population cultured under hypoxic conditions as compared to a cell population cultured under non-hypoxic conditions. In another embodiment, the detectable EPO expression is more in a cell population cultured via perfusion than in a cell population not cultured via perfusion. In additional embodiments, the detectable EPO expression is more in a cell population cultured under hypoxic conditions than in a cell population cultured under non-hypoxic conditions. In certain other embodiments, the induction of EPO expression and / or the increased EPO expression can be observed at a level of induction and / or increased expression of less than about 5% oxygen, i.e., when culturing the cells under hypoxic culture conditions, as compared to cells cultured under atmospheric oxygen levels (about 21%), i.e., non-hypoxic culture conditions. In another embodiment, the increased EPO expression can be observed when culturing fewer cells than under 21% oxygen (in air) conditions. In yet another aspect, the present invention provides a transplantable construct containing one or more cell populations described herein. In one embodiment, the present invention provides a transplantable construct for providing a cell population that produces erythropoietin (EPO) to a subject in need thereof.

[0050] for use, the construct comprising: a) a scaffold containing one or more biocompatible synthetic polymers or naturally occurring proteins or peptides, and b) a first cell population enriched for mammalian cells deposited within or on the surface of the scaffold that produce EPO. In another embodiment, the invention provides a transplantable construct for providing a cell population that produces erythropoietin (EPO) to a subject in need thereof, the construct comprising: a) a porous scaffold containing one or more biocompatible synthetic polymers or naturally occurring proteins or peptides, and b) a mixture of cells containing: i) a first cell population enriched for mammalian cells that produce EPO, and ii) a second cell population enriched for cells that do not produce EPO, the mixture of cells being deposited on the surface of and / or within the pores of the scaffold. In some embodiments, EPO expression is greater in the first cell population compared to the second cell population. In certain other embodiments, the second cell population can contain one or more kidney-derived cell types including, but not limited to, one or more of the following:

[0051] tubule-derived cells, glomerulus-derived cells, stroma-derived cells, connective tissue-derived cells, collecting duct-derived cells, blood-derived cells, or blood vessel-derived cells. In another embodiment, the second cell population is enriched for kidney tubule cells. In some embodiments of the invention, there is provided a population enriched for kidney tubule cells characterized by the expression of one or more of the following, but not limited to: megalin, cubilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2. In another embodiment, the invention provides a transplantable construct for providing a cell population that produces erythropoietin (EPO) to a subject in need thereof, the construct comprising: a) a porous scaffold containing one or more biocompatible synthetic polymers or naturally occurring proteins or peptides, and b) a mixture of cells containing: i) a first cell population enriched for mammalian cells that produce EPO, and ii) a second cell population enriched for cells that do not produce EPO, the mixture of cells being deposited on the surface of and / or within the pores of the scaffold. In some embodiments, EPO expression is greater in the first cell population compared to the second cell population. In certain other embodiments, the second cell population can contain one or more kidney-derived cell types including, but not limited to, one or more of the following: tubule-derived cells, glomerulus-derived cells, stroma-derived cells, connective tissue-derived cells, collecting duct-derived cells, blood-derived cells, or blood vessel-derived cells. In another embodiment, the second cell population is enriched for kidney tubule cells. In some embodiments of the invention, there is provided a population enriched for kidney tubule cells characterized by the expression of one or more of the following, but not limited to: megalin, cubilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2. In some embodiments, EPO expression is greater in the first cell population compared to the second cell population. In certain other embodiments, the second cell population can contain one or more kidney-derived cell types including, but not limited to, one or more of the following: tubule-derived cells, glomerulus-derived cells, stroma-derived cells, connective tissue-derived cells, collecting duct-derived cells, blood-derived cells, or blood vessel-derived cells. In another embodiment, the second cell population is enriched for kidney tubule cells. In some embodiments of the invention, there is provided a population enriched for kidney tubule cells characterized by the expression of one or more of the following, but not limited to: megalin, cubilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2. tubule-derived cells, glomerulus-derived cells, stroma-derived cells, connective tissue-derived cells, collecting duct-derived cells, blood-derived cells, or blood vessel-derived cells. In another embodiment, the second cell population is enriched for kidney tubule cells. In some embodiments of the invention, there is provided a population enriched for kidney tubule cells characterized by the expression of one or more of the following, but not limited to: megalin, cubilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2. In some embodiments, EPO expression is greater in the first cell population compared to the second cell population. In certain other embodiments, the second cell population can contain one or more kidney-derived cell types including, but not limited to, one or more of the following: tubule-derived cells, glomerulus-derived cells, stroma-derived cells, connective tissue-derived cells, collecting duct-derived cells, blood-derived cells, or blood vessel-derived cells. In another embodiment, the second cell population is enriched for kidney tubule cells. In some embodiments of the invention, there is provided a population enriched for kidney tubule cells characterized by the expression of one or more of the following, but not limited to: megalin, cubilin, N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2. tubule-derived cells, glomerulus-derived cells, stroma-derived cells, connective tissue-derived cells, collecting duct-derived cells, blood-derived cells, or blood vessel-derived cells. In another embodiment, the second cell population is enriched for kidney tubule cells. In some embodiments of the invention, there is provided a population enriched for kidney tubule cells characterized by the expression of one or more of the following, but not limited to:

[0052] In one embodiment, the mixture of kidney cells provided by the present invention can include a cell population derived from the type of source of kidney tissue, as described herein. In other embodiments, the first cell population and the second cell population are derived from kidney tissue or cultured kidney cells. In another embodiment, the first cell population contains a greater proportion of cells that produce erythropoietin (EPO) than a non-concentrated population of mammalian cells that produce EPO. In another embodiment, the first cell population contains glomerular cells and vascular cells in addition to cells that produce EPO. In yet another embodiment, the first cell population contains a greater proportion of cells that produce EPO than the second cell population. A further embodiment of the present invention includes a first cell population that contains a smaller proportion of kidney tubular cells than a non-concentrated population of mammalian cells that produce erythropoietin (EPO). In other embodiments, the first cell population contains a smaller proportion of kidney tubular cells than the second cell population. In some embodiments, the cells are combined with a biomaterial. In one embodiment, the scaffold or biomaterial is configured as a three-dimensional (3-D) porous scaffold. In another embodiment, the 3-D porous scaffold is suitable for the capture or attachment of mammalian cells. In yet another embodiment, the scaffold or biomaterial is configured as a liquid or semi-fluid gel suitable for embedding, attaching, or coating mammalian cells. In one embodiment, the cell population suitable for use in the structures of the present invention is derived from an autologous or allogeneic kidney sample. In certain other embodiments, the sample is a kidney biopsy.

[0053] In another aspect, the present invention provides a method of treating a subject in need of a cell population enriched for cells that produce EPO. In one embodiment, the method is for treating erythropoietin (EPO) deficiency in a subject in need thereof, and comprises administering to the subject a composition containing a first cell population enriched for mammalian cells that produce EPO. In another embodiment, the first cell population is enriched for cells that produce EPO, glomerular cells, and vascular cells. In one embodiment, the EPO deficiency is anemia. In another embodiment, the EPO deficiency or anemia is secondary to renal insufficiency in the subject. In certain other embodiments, the EPO deficiency or anemia is secondary to a disease selected from the group consisting of chronic renal insufficiency, primary EPO deficiency, chemotherapy or antiviral therapy, non-myeloid cancer, HIV infection, liver disease, heart failure, rheumatoid arthritis, or multiple organ system failure. In another embodiment, the method is for treating a kidney disease in a subject in need thereof, and comprises administering to the subject a composition containing a first cell population enriched for mammalian cells that produce EPO. In another embodiment, the first cell population is enriched for cells that produce EPO, glomerular cells, and vascular cells. In some embodiments, the composition administered to the subject in need further contains a second kidney cell population that is not enriched for cells that produce EPO. In other embodiments, the composition further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population is on the surface of the scaffold. In another embodiment, the EPO deficiency or anemia is secondary to renal insufficiency in the subject. In certain other embodiments, the EPO deficiency or anemia is secondary to a disease selected from the group consisting of chronic renal insufficiency, primary EPO deficiency, chemotherapy or antiviral therapy, non-myeloid cancer, HIV infection, liver disease, heart failure, rheumatoid arthritis, or multiple organ system failure. In another embodiment, the method is for treating a kidney disease in a subject in need thereof, and comprises administering to the subject a composition containing a first cell population enriched for mammalian cells that produce EPO. In another embodiment, the first cell population is enriched for cells that produce EPO, glomerular cells, and vascular cells.

[0054] In another embodiment, the method is for treating a kidney disease in a subject in need thereof, and comprises administering to the subject a composition containing a first cell population enriched for mammalian cells that produce EPO. In another embodiment, the first cell population is enriched for cells that produce EPO, glomerular cells, and vascular cells. In some embodiments, the composition administered to the subject in need further contains a second kidney cell population that is not enriched for cells that produce EPO. In other embodiments, the composition further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population is on the surface of the scaffold.

[0055] In some embodiments, the composition administered to the subject in need further contains a second kidney cell population that is not enriched for cells that produce EPO. In other embodiments, the composition further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population is on the surface of the scaffold. In some embodiments, the composition administered to the subject in need further contains a second kidney cell population that is not enriched for cells that produce EPO. In other embodiments, the composition further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population is on the surface of the scaffold. In some embodiments, the composition administered to the subject in need further contains a second kidney cell population that is not enriched for cells that produce EPO. In other embodiments, the composition further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population is on the surface of the scaffold. In some embodiments, the composition administered to the subject in need further contains a second kidney cell population that is not enriched for cells that produce EPO. In other embodiments, the composition further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population is on the surface of the scaffold. and / or deposited within the pores. In additional embodiments, the composition contains one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides and further includes a porous scaffold, and the first cell population and the second cell population are deposited on the surface of the scaffold and / or within the pores. In another embodiment, the first cell population and / or the second cell population is derived from mammalian kidney tissue or cultured mammalian kidney cells. In other embodiments, the first cell population and / or the second cell population is derived from autologous or allogeneic kidney samples. In one embodiment, the sample is a kidney biopsy. In one embodiment, the second cell population is enriched for kidney tubule cells. In another embodiment, the kidney tubule cells can include, but are not limited to, expression of one or more of the following tubular cell markers: megalin, cubilin , N-cadherin, E-cadherin, aquaporin-1, and aquaporin-2. In all embodiments, the second cell population includes one or more kidney-derived cell types selected from the group consisting of tubular-derived cells, glomerular-derived cells,

[0056] interstitial-derived cells, collecting duct-derived cells, connective tissue-derived cells, blood-derived cells, or vascular-derived cells. In all embodiments, the second cell population is a heterogeneous, unfractionated population or is relatively depleted of glomerular cells, vascular cells, and cells that produce oxygen-responsive EPO compared to the first cell population. In another aspect, the invention includes a method of providing erythrocyte homeostasis to a subject in need thereof.

[0057]

[0058]

[0058] In one embodiment, the method comprises: a) administering to a subject a composition containing a population of cells enriched for mammalian cells that produce EPO, and b) measuring in a test sample from the subject that the level of an erythropoiesis function indicator is different from the level of a control indicator, wherein the difference in the indicator levels indicates erythrocyte homeostasis in the subject. 1 cell population containing the The level of the control indicator is measured in a test sample from the subject, and the difference in the indicator levels indicates erythrocyte homeostasis in the subject. Steps, the difference in indicator levels indicates erythrocyte homeostasis in the subject.

[0059] In another embodiment, the method comprises: a) administering to a subject a composition comprising a first population of cells enriched for mammalian cells that produce EPO and a second population of cells not enriched for cells that produce EPO, and b) measuring in a test sample from the subject that the level of an erythropoiesis function indicator is different from the level of a control indicator, wherein the difference in the indicator levels indicates erythrocyte homeostasis in the subject. A first cell population enriched for mammalian cells that produce EPO and a second cell population not enriched for cells that produce EPO, and Administering the composition to the subject, and b) measuring in a test sample from the subject that the level of an erythropoiesis function indicator is different from the level of a control indicator, wherein the difference in the indicator levels indicates erythrocyte homeostasis in the subject. The level of the control indicator is measured in a test sample from the subject, and the difference in the indicator levels indicates erythrocyte homeostasis in the subject. Steps, the difference in indicator levels indicates erythrocyte homeostasis in the subject.

[0060] In another aspect, the present invention includes a method for improving the renal function of a subject in need thereof. In certain other In embodiments, the method comprises: a) administering to a subject a composition containing a first population of cells enriched for mammalian cells that produce EPO, and b) measuring in a test sample from the subject that the level of a renal function indicator is different from the level of a control indicator, wherein the difference in the indicator levels indicates an improvement in renal function in the subject. In another embodiment, the composition A first cell population enriched for mammalian cells that produce EPO, and The level of the control indicator is measured in a test sample from the subject, and the difference in the indicator levels indicates an improvement in renal function in the subject. Steps, the difference in indicator levels indicates an improvement in renal function in the subject. In another embodiment, the composition Further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population is deposited on the surface and / or In the pores of the scaffold. Or within the pores.

[0061] In another embodiment, the method comprises: a) administering to a subject a composition enriched for mammalian cells that produce EPO containing a first cell population and a second cell population that is not enriched for cells that produce EPO administering the composition to a subject, and b) measuring in a test sample from the subject whether the level of an indicator of renal function is different from the indicator level of a control, wherein the difference in indicator levels indicates improved renal function in the subject. In another embodiment, the composition further comprises a porous scaffold containing one or more biocompatible synthetic polymers and / or naturally occurring proteins or peptides, and the first cell population and the second cell population are deposited on the surface of and / or within the pores of the scaffold.

[0062] In other embodiments, the first cell population and / or the second cell population are derived from mammalian kidney tissue or cultured mammalian kidney cells. In another embodiment, the first cell population and / or the second cell population are derived from a self or non-self kidney sample. In certain other embodiments, the sample is a kidney biopsy.

[0063] In one embodiment, the first cell population is enriched for cells that produce hypoxia-responsive EPO. In another embodiment, the first cell population is enriched for cells that produce hypoxia-responsive epo as well as glomerular cells and vascular cells.

[0064] In one embodiment, the second cell population is enriched for kidney tubular cells. In another embodiment, the kidney tubular cells can comprise one or more of the following, but are not limited thereto, and are characterized by the expression of tubular cell markers; hyaluronan synthase 2 (HAS2), CYP2D25 (vitamin D3 25-hydroxylase), megalin ​​​​​, Cubilin, N-cadherin, E-cadherin, aquaporin-1, aquaporin-2 and aquaporin-4. In another embodiment, the second cell population is enriched for kidney tubule cells and contains epithelial cells of the collecting duct. In another embodiment, the second cell set is relatively enriched for tubule cells, contains collecting duct epithelial cells, and relatively reduced in cells that produce hypoxia-responsive EPO, glomerular cells, and vascular cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0065]

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Mode for Carrying Out the Invention

[0066] The present invention relates to isolated kidney cells comprising renal tubules and kidney cells that produce erythropoietin (EPO), a method for isolating and culturing the same, and a method for treating a subject in need thereof with bioactive kidney cells comprising a concentrated population of renal tubules and cells that produce EPO. defined Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. (RLanza, R Langer, & J Vacanti eds.) Principles of Tissue Engineering, 3rd Ed., 2007 provides general guidance to one of ordinary skill in the art for many of the terms used in this application. One of ordinary skill in the art will understand many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. In fact, the present invention is in no way limited to the methods and materials described. As used herein, "cell population" generally refers to a plurality of cells obtained by direct isolation from a suitable tissue source from a mammal. The isolated cell population can be subsequently cultured in vitro. One of ordinary skill in the art will understand the various methods for isolating and culturing cell populations for use with the present invention and the various numbers of cells in a cell population suitable for use in the present invention. A cell population can be an unfractionated, heterogeneous cell population derived from the kidney. For example, a heterogeneous cell population can be isolated from a renal biopsy or from whole kidney tissue. Alternatively, a heterogeneous cell population can be

[0067] An unfractionated heterogeneous cell population can also be referred to as an unconcentrated cell population.

[0068] It may be derived from in vitro culture of mammalian cells established from kidney biopsy or whole kidney tissue.

[0069] ​​​​​​​​​ As used herein, the term "mixture" refers to a combination of two or more isolated, enriched cell populations arising from an unfractionated, heterogeneous cell population. According to certain embodiments, the cell populations of the present invention are cell populations of the kidney. An "enriched" cell population or preparation refers to a cell population arising from a starting kidney cell population (e.g., an unfractionated, heterogeneous cell population) that contains a greater proportion of that cell type than the proportion of that particular cell type in the starting population. For example, a starting kidney cell population can be enriched for a first, second, third, fourth, fifth, etc., target cell population. As used herein, the terms "cell population," "cell preparation," and "cell prototype" are used interchangeably.

[0070]

[0071] In one aspect, an "enriched" cell population as used herein refers to a cell population arising from a starting kidney cell population (e.g., a cell suspension from a renal biopsy or cultured mammalian kidney cells) that contains a greater proportion of cells capable of producing EPO than the proportion of cells capable of producing EPO in the starting population. For example, "B4" is a cell population arising from a starting kidney cell population that contains a greater proportion of cells producing EPO, glomerular cells, and vascular cells compared to the starting population. The cell populations of the present invention can be enriched for one or more cell types and depleted of one or more other cell types. For example, a cell population enriched for EPO-producing cells can be enriched for interstitial fibroblasts and depleted of tubular cells and collecting duct epithelial cells compared to the interstitial fibroblasts and tubular cells of the starting cell population from which the enriched cell population is derived. An EPO-enriched cell population ​​​​​​​​​​​​​​​In all embodiments that refer to the "B4" population, the enriched cell population is endogenous a heterogeneous population of cells containing cells that can produce EPO in an oxygen-regulated manner, as indicated by oxygen-regulated EPO expression from the native EPO gene.

[0072] In another aspect, the enriched cell population can also refer to a cell population arising from the above-described starting kidney cell population that contains a greater proportion of cells expressing one or more tubule cell markers than the proportion of cells expressing one or more tubule cell markers in the starting population. For example, "B2" refers to a cell population arising from a starting kidney cell population that contains a greater proportion of tubule cells compared to the starting population. Further, a cell population enriched for cells expressing one or more tubule cell markers (or "B 2") can contain some epithelial cells from the collecting duct system. A cell population enriched for cells expressing one or more tubule cell markers (or "B2") has relatively fewer EPO-producing cells, glomerular cells, and vascular cells removed, but the enriched population can contain a smaller proportion of these cells (EPO-producing, glomerular, and vascular) compared to the starting population. Generally, a heterogeneous cell population has one or more cell types removed such that the removed cell population contains a smaller proportion of cell types compared to the proportion of cell types contained in the heterogeneous cell population prior to removal. The cell types that can be removed ​In an embodiment, cell types that can be removed include low-granularity and low-viability debris and small cells having a density greater than about 1.0 95 g / mL, including "B5". In some embodiments, a cell population enriched for tubular cells has all of the following removed relatively completely: "B1", "B5", cells expressing oxygen-regulated EPO, glomerular cells , and vascular cells.

[0073] As used herein, the term "hypoxic" culture conditions refers to culture conditions for cells in which the available oxygen level in the culture system is reduced compared to standard culture conditions in which the cells are cultured at the oxygen level in air (about 21%). Non-hypoxic conditions are referred to herein as normal or normoxic culture conditions .

[0074] As used herein, the term "oxygen regulatable" refers to the ability of a cell to regulate gene expression (increase or decrease) based on the amount of oxygen available to the cell. "Hypoxia inducible" refers to upregulation of gene expression in response to a decrease in the partial pressure of oxygen ( regardless of pre-induction or onset of the partial pressure of oxygen).

[0075] As used herein, "biomaterial" refers to natural or synthetic biocompatible materials suitable for introduction into living tissue. Natural biomaterials are materials made by living systems. Synthetic biomaterials are materials not made by living systems. The biomaterials disclosed herein can be a combination of natural and synthetic biocompatible materials. Biomaterials used herein include, for example, polymer-matrices and scaffolds. One of ordinary skill in the art will appreciate that biomaterials can be configured in a variety of forms, such as a liquid hydrogel suspension, a porous foam, and can be 1 It will be understood that it can include one or more natural or synthetic biocompatible materials.

[0076] As used herein, the term "anemia" refers to the inappropriate production of functional EPO protein by cells that produce EPO in a subject, and / or the inappropriate release of EPO protein into the systemic circulation, and / or the unresponsiveness of erythroblasts in the bone marrow to EPO protein, resulting in a deficiency in the number of red blood cells and / or hemoglobin levels. Anemic subjects are unable to maintain erythrocyte homeostasis. Generally, anemia is associated with a decrease or loss of kidney function (e.g., chronic renal failure), anemia associated with related EPO deficiency, anemia associated with congestive heart failure, anemia associated with myelosuppressive treatments such as chemotherapy or antiviral therapy (e.g., AZT), anemia associated with non-myeloid cancers, anemia associated with viral infections such as HIV, and

[0077] anemia in chronic diseases such as autoimmune diseases (e.g., rheumatoid arthritis), liver diseases, and multi-organ system failure.

[0078] As used herein, the term "kidney disease" refers to any stage or degree of acute or chronic renal insufficiency that causes a deficiency in the ability of the kidneys to perform the functions of blood filtration and the removal of excess fluid, electrolytes, and waste products of the blood. Kidney diseases also include endocrine dysfunctions such as anemia (EPO deficiency) and mineral imbalances (vitamin D can be secondary to a variety of conditions including, but not limited to, liver diseases.

[0079] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures for kidney disease, anemia, EPO deficiency, lack of tubular transport, or lack of glomerular filtration, with the goal of reversing, preventing, or delaying (reducing) the target disease. Those in need of treatment are those who already have kidney disease, anemia, EPO deficiency, lack of tubular transport, or lack of glomerular filtration, as well as those who tend to have kidney disease, anemia, EPO deficiency, lack of tubular transport, or lack of glomerular filtration, or those in whom kidney disease, anemia, EPO deficiency, lack of tubular transport, or lack of glomerular filtration is prevented. The term "treatment" as used herein includes stabilization and / or improvement of renal function.

[0080] The term "construct" refers to one or more cell populations deposited on or within a scaffold or matrix composed of one or more synthetic or naturally occurring biocompatible materials. One or more cell populations can be coated with, deposited on, embedded within, attached to, or captured within a biomaterial composed of one or more synthetic or naturally occurring biocompatible polymers, proteins, or peptides. One or more cell populations can be combined with the biomaterial or scaffold or matrix in vitro and in vivo. Generally, the one or more biocompatible materials used to form the scaffold / biomaterial are selected to direct, facilitate, or enable the formation of the organization of at least one multicellular, three-dimensional, cell population deposited thereon. To generate a construct, use ​​​​​​​​​​​​​ One or more biomaterials used are for the dispersion and / or integration or engraftment of the construct or for directing, facilitating, or enabling the cellular components of the construct that include tissue or for directing, facilitating, or enabling the survival, engraftment, tolerance, or functional ability of the construct or the cellular components of the construct.

[0081] The term "subject" shall mean any single human subject who is experiencing or has experienced one or more signs, symptoms, or other indicators of a kidney disease, anemia, or EPO deficiency that is suitable for treatment. Such subjects include, but are not limited to, subjects who have been newly diagnosed with, previously diagnosed with and currently experiencing a relapse or recurrence of, or at risk of, a kidney disease, anemia, or EPO deficiency, regardless of cause. The subject may or may not have been previously treated for a kidney disease, anemia, or EPO deficiency.

[0082] The term "patient" shall mean any single animal, more preferably a mammalian animal (including non-human animals such as dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates, etc.), for which treatment is desired. More preferably, the patient herein is a human.

[0083] The term "sample", or "patient sample", or "biological sample" shall generally mean any biological sample obtained from a subject or patient, body fluid, body tissue, cell line, tissue culture, or other source. This term includes, for example, tissue biopsies such as kidney biopsies. 。This term includes, for example, cultured cells such as cultured mammalian kidney cells. Mammals Methods for obtaining tissue biopsies and cultured cells from mammals are well known in the art When the term "sample" is used alone, it is still meant that the "sample" is a "biological sample" or "patient sample", i.e., these terms are used interchangeably .

[0084] The term "test sample" refers to a sample from a subject that has been processed by the method of the present invention Test samples include, but are not limited to, blood, semen, serum, urine, bone marrow, mucosa, tissue, etc., and may originate from various sources in mammalian subjects .

[0085] The term "control" or "control sample" refers to a negative or positive control that is predicted to assist in the mutual association of negative or positive results with the results of the test sample Controls suitable for the present invention include samples known to exhibit indicators characteristic of normal erythrocyte homeostasis, samples known to exhibit indicators characteristic of anemia, samples obtained from subjects known not to have anemia, and samples obtained from subjects known to have anemia, but are not limited to these Further controls suitable for use in the method of the present invention include samples derived from subjects who have been treated with pharmacological agents known to regulate erythropoiesis (e.g., recombinant EPO or EPO analogs), but are not limited to these Furthermore, the control may be a sample obtained from a subject prior to treatment by the method of the present invention A further suitable control is a test sample obtained from a subject known to have any type or stage of kidney disease, as well as any type or stage of kidney disease PO analogs), and samples obtained from subjects who have been treated with pharmacological agents known to regulate erythropoiesis (e.g., recombinant EPO or EPO analogs), but are not limited to these Furthermore, the control may be a sample obtained from a subject prior to treatment by the method of the present invention A further suitable control is a test sample obtained from a subject known to have any type or stage of kidney disease, as well as any type or stage of kidney disease known to have, and test samples obtained from subjects known to have any type or stage of kidney disease known to have, and test samples obtained from subjects known to have any type or stage of kidney disease It may also be a sample from a subject known not to have it. The control may be a healthy control or the like. Those skilled in the art will understand other subjects suitable for use in the present invention.

[0086] Cell population The present invention provides an isolated, heterogeneous population of renal cells and mixtures thereof that are enriched for a specific bioactive component or cell type and / or depleted of specific inactive or undesired components or cell types for use in the treatment of kidney diseases, i.e., providing stabilization and / or improvement and / or regeneration of kidney function.

[0087] Cell population exhibiting bioactivity In one aspect, the present invention is based on the unexpected discovery that a specific subfraction of a heterogeneous population of renal cells enriched for a bioactive component and depleted of inactive or undesired components results in better therapeutic and regenerative results than the starting population. For example, the bioactive components of the present invention from which inactive or undesired components, such as B1 and B5, have been removed such as B2, B4, and B3, alone or in combination, result in unexpected stabilization and / or improvement and / or regeneration of kidney function. In a preferred embodiment, the cell population exhibiting bioactivity is B2. In certain embodiments, the B2 cell population is mixed with B4 and in other embodiments, the B2 cell population is mixed with B3. The B2 cell population is megalin, cubilin, hyaluronan synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad),

[0088] E-cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aq p2), RAB17, member-RAS oncogene family- (Rab17), GATA binding protein 3 (Gata3), ion transport regulator-4 containing FXYD domain (F xyd4), solute carrier family-9 (sodium / hydrogen exchanger), member-4 (Slc 9a4), aldehyde dehydrogenase 3 family-, member-B1 (Aldh3b1), al dehyde dehydrogenase 1 family-, member-A3 (Aldh1a3), and calpain- 8 (Capn8) selected from the group consisting of tubular cell markers, and collecting duct markers characterized by the expression of aquaporin-4 (Aqp4), greater than B3 and / or B4 and more granular, thus having a buoyant density of about 1.045 g / mL to about 1.063 g / mL (rodents), about 1.045 g / mL to 1.052 g / mL (human), and about 1.045 g / mL to about 1.058 g / mL (dog).

[0089] The B3 cell population is characterized by the expression of vascular markers, glomerular markers, and proximal tubular markers, and cells that produce some EPO, and is of medium size and granular compared to B2 and B4, thus having a buoyant density of about 1.063 g / mL to about 1.073 g / mL (rodents), about 1.052 g / mL to 1.063 g / mL (human), and about 1.058 g / mL to about 1.063 g / mL (dog). B3 is characterized by the expression of a marker selected from the group consisting of one or more of the following: aquaporin 7 (Aqp7) , ion transport regulator-2 containing FXYD domain (Fxyd2), solute carrier family ​Milli-17 (sodium phosphate), member-3 (Slc17a3), solute carrier family -3, member-1 (Slc3a1), claudin 2 (Cldn2), napsin A aspartic protease (Napsa), solute carrier family-2 (facilitated glucose transport body), member-2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep) , transmembrane protein 27 (Tmem27), acyl-CoA synthetase medium-chain family member -2 (Acsm2), glutathione peroxidase 3 (Gpx3), fructose-1 ,6-bisphosphatase 1 (Fbp1), and alanine aminotransferase 2 (Agxt2). B3 is also characterized by the vascular expression marker platelet endothelial cell adhesion molecule (Pecam) and the glomerular expression marker podocin (Podn).

[0090] The B4 cell population is characterized by a vascular marker set containing one or more of PECAM, VEGF, KDR, HIF1a, a glomerular marker set containing one or more of podocin (Podn) and nephrin (Neph), and an oxygen-regulated EPO-enriched population compared to unfractionated (UNFX), B2, and B3. B4 is also characterized by the expression of one or more of the following markers: chemokine (C-X-C motif) receptor 4 (Cxcr4), endothelin receptor type B (Ednrb), collagen, type V, alpha 2 (Col5a2), cadherin 5 (Cdh5), plasminogen activator , tissue (Plat), angiopoietin 2 (Angpt2), kinase insert domain receptor (Kdr), secreted protein, acidic, cysteine-rich (osteonectin ), ​​​Cutin)(Sparc), Serglycin (Srgn), TIMP metallopeptidase inhibitor Agent 3 (Timp3), Wilms tumor 1 (Wt1), Featherless type MMTV integration site family Member-, member-4 (Wnt4), regulator of G-protein signaling 4 (Rgs4) , Platelet endothelial cell adhesion molecule (Pecam), and Erythropoietin (Epo). B4 is , Characterized by being smaller and less granular compared to either B2 or B3 and having a buoyant density of about 1.073 g / mL to about 1.091 g / mL (rodents), about 1.063 g / mL to about 1.091 g / mL (humans and dogs).

[0091] Hyaluronic acid production by B2 and B4 Hyaluronan (also referred to as hyaluronic acid or hyaluronate) is a glycosaminogly can (GAG), composed of regular repeating sequences of non-sulfated disaccharide units, particularly N-acetylglucosamine and gluc uronic acid. Its molecular weight can range from 400 daltons (disaccharide) to over 1 million daltons. It is found in varying amounts in all tissues such as adult skin, cartilage, and eyes, as well as in most body fluids, if not all. It is abundantly present in the early embryo. The space formed by hyaluronan, actually by GAGs in general, allows hyaluronan to play roles in cell migration, cell adhesion, wound repair, organogenesis, adhesion of immune cells, activation of intracellular sig nal transduction, and tumor metastasis. These roles are mediated by specific proteins and proteoglycans that bind to hyaluronan . Cell motility and adhesion of immune cells are mediated by the cell surface receptor RHAMM (R eceptor for Hyaluronal-Mediated Motility ​​; mediated by Hardwick et al., 1992) and CD44 (Jalkenan e t al., 1987; Miyake et al., 1990). Hyaluronic acid is synthesized directly in the inner membrane of the cell surface, and the growing polymer is pushed out of the cell through the membrane once it is synthesized. Synthesis is mediated by a single protein enzyme, hyaluronan synthase (HAS), which consists of at least three members from a gene family. of.

[0092] Hyaluronic acid interacts with CD44, and such interaction can mobilize very resident cells (such as mesenchymal stem cells (MSCs)) in damaged kidney tissue among other actions and promote kidney regeneration (Kidney International (2007) 72, 430 - 441), which has recently been revealed. of.

[0093] Surprisingly, B2 and B4 cell preparations can express high - molecular - weight species of hyaluronic acid (HA) both in vitro and in vivo through the action of hyaluronan synthase - 2 (HAS - 2), a marker that is more specifically concentrated in the B2 cell population . Treatment with B2 in the 5 / 6Nx model was shown to reduce fibrosis simultaneously with strong in - vivo HAS - 2 expression and the expected production of high - molecular - weight HA in the treated tissue. In particular, the untreated 5 / 6Nx model developed fibrosis with only slight detection of HAS - 2, and little high - molecular - weight HA was produced ., . Without being bound by theory, this anti - inflammatory high - molecular - weight species of HA, mainly produced by B2 (and to some extent by B4), is responsible for the reduction of renal fibrosis and kidney regeneration ., ., ., ., ., A hypothesis is proposed that it acts synergistically with the cell preparation in the assistance. Therefore, the present invention includes the delivery of the prototype of the cells of the present invention in a biomaterial containing hyaluronic acid. Also, the present invention also contemplates providing a biomaterial component for regenerative stimulation through direct production by transplanted cells or stimulation of production. is contemplated by the present invention.

[0094] In one aspect, the present invention provides an isolated heterogeneous population of EPO-producing renal cells for use in the treatment of kidney diseases, anemia, and / or EPO deficiency in a subject in need thereof. In one embodiment, the cell population is derived from a renal biopsy. In another embodiment, the cell population is derived from whole kidney tissue. In certain other embodiments, the cell population is derived from in vitro culture of mammalian renal cells established from a renal biopsy or whole kidney tissue. In all embodiments, these populations are unfractionated cell populations, also referred to herein as non-concentrated cell populations. In all embodiments, these populations are unfractionated cell populations and are not concentrated herein and are also referred to as non-concentrated cell populations.

[0095] In another aspect, the present invention provides an isolated population of further concentrated erythropoietin (EPO)-producing renal cells such that the ratio of cells producing EPO in the concentrated subpopulation is higher compared to the ratio of cells producing EPO in the starting or initial cell population. In one embodiment, the concentrated cell fraction producing EPO contains a higher proportion of interstitial fibroblasts and a lower proportion of tubular cells compared to the interstitial fibroblasts and tubular cells contained in the non-concentrated initial population. In certain embodiments, the concentrated cell fraction producing EPO contains a higher proportion of glomerular cells and vascular cells compared to the glomerular cells, vascular cells, and collecting duct cells contained in the non-concentrated initial population. In certain embodiments, the concentrated cell fraction producing EPO contains a higher proportion of glomerular cells and vascular cells compared to the glomerular cells contained in the non-concentrated initial population, and contain a lower proportion of collecting duct cells. In such embodiments, these populations are referred to herein as "B4" cell populations.

[0096] In another aspect, the invention provides an EPO-producing renal cell population mixed with one or more additional renal cell populations. In one embodiment, the cell population that produces EPO is a first cell population that is enriched for cells that produce EPO, such as B4. In another embodiment, the cell population that produces EPO is a first cell population that is not enriched for cells that produce EPO, such as B2. In another embodiment, the first cell population is mixed with a second renal cell population. In some embodiments, the second cell population is enriched for tubular cells and can be indicated by the presence of the phenotype of tubular cells. In another embodiment, the phenotype of tubular cells can be indicated by the presence of a tubular cell marker. In another embodiment, the phenotype of tubular cells can be indicated by the presence of one or more tubular cell markers. Tubular cell markers include megalin, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E- cadherin (Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp 2), RAB17, member-RAS oncogene family (Rab17), GATA-binding protein Aldehyde dehydrogenase 1 family, member A3 (Aldh1a3), and calpain-8 (Capn8), among others. In another embodiment, the first cell population comprises, but is not limited to, stroma-derived cells, tubule cells, collecting duct-derived cells, glomerulus-derived cells, and / or blood or vessel-derived cells, and is mixed with at least one of several types of kidney cells including, but not limited to, these.

[0097] In one aspect, the EPO-producing kidney cell population of the present invention is characterized by a biological response to EPO expression and oxygen such that a decrease in the oxygen partial pressure of the culture system results in induction in the expression of EPO. In one embodiment, the cell population producing EPO is enriched relative to cells producing EPO. In one embodiment, the cell population is cultured under conditions where the cells are exposed to a decrease in the oxygen level available in the culture system compared to a cell population cultured with oxygen available at standard atmospheric levels (about 21%), such that the expression of EPO is induced. In one embodiment, cells producing EPO cultured under lower oxygen conditions express higher levels of EPO compared to cells producing EPO cultured under standard oxygen conditions. Generally, culturing cells at lower available oxygen levels (also referred to as hypoxic culture conditions) means that the reduced oxygen level is lower compared to cell culture with oxygen available at standard atmospheric levels (also referred to as standard or normoxic culture conditions). In one embodiment, hypoxic cell culture conditions include culturing the cells with less than about 1% oxygen, less than about 2% oxygen, less than about 3% oxygen, less than about 4% oxygen, or less than about 5% oxygen. In another embodiment, standard or normoxic culture conditions are about 10% oxygen, about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen, or about 20% oxygen. of oxygen, about 14% oxygen, about 15% oxygen, about 16% oxygen, about 17% oxygen, about 18% Cultivating cells at about 10% oxygen, about 19% oxygen, about 20% oxygen, or about 21% oxygen. Includes.

[0098] In certain other embodiments, cells are cultured with less than about 5% available oxygen and expression of EPO is improved. By comparing the current levels of EPO to cells cultured in atmospheric (approximately 21%) oxygen, Induction or increased expression can be obtained and observed. Induction occurs when a culture of cells is grown in atmospheric oxygen (approximately 21%) for some period of time. In the first culture step, the available oxygen level is reduced so that the cells grow to less than about 5% of the available oxygen. and a second culture step in which the cells are cultured in an oxygen-rich environment. In another embodiment, the method is performed in a culture of cells that are responsive to hypoxic conditions. The expression of certain EPOs is regulated by HIF1α. Other oxygen-manipulating culture conditions that are known may be used with the cells described herein. You will understand that.

[0099] In one embodiment, the enriched population of EPO-producing mammalian cells is a biomarker for the response to perfusion conditions. In one embodiment, the perfusion conditions are characterized by transient responsiveness (e.g., expression of EPO). In one embodiment, the cells are cultured in a culture medium that includes continuous, intermittent, or continuous fluid flow (perfusion). The medium is circulated or recirculated intermittently or continuously in a manner that transfers power to the cells via flow. In one embodiment, the expression of EPO is induced transiently when the cells are heated or stirred. Cells exposed to continuous, intermittent, or continuous fluid flow exhibit the fre- quency required for the formation of three-dimensional structures. - scaffolds and / or spaces, such three-dimensional structures in or on the material and are cultured in a manner in which they are represented. In one embodiment, the cells are porous beads cultured on and exposed to intermittent or continuous fluid flow by a rocking platform, an orbital platform, or a spinner flask . In another embodiment, the cells are cultured on a three-dimensional scaffold and placed within a device, whereby the scaffold is fixed and fluid flows in one direction through or across the scaffold. Those skilled in the art will understand that other perfusion culture conditions known in the art can be used for the cells described herein.

[0100] Inactive cell population As described herein, the present invention is based in part on the unexpected discovery that a particular subfraction of a heterogeneous population of renal cells, enriched for bioactive components and depleted of inactive or undesired components, provides superior therapeutic and regenerative outcomes compared to the starting population. In a preferred embodiment, the cell population of the present invention has the B1 and / or B5 cell populations removed.

[0101] The B1 cell population includes the large granular cells of the collecting ducts and the renal tubule system, and the cells of the population have a buoyant density of less than about 1.045 g / mL. The B5 cell population is composed of debris and small cells with low particle size and viability and has a buoyant density of greater than about 1.091 g / mL.

[0102] Method for isolating and culturing cell population In one aspect, the present invention provides a renal cell component, e.g., a concentrated for therapeutic use including the treatment of kidney diseases, anemia, EPO deficiency, renal tubular transport deficiency, and glomerular filtration deficiency Provided is a method for separating and isolating a cell population. In one embodiment, the cell population is , newly digested, i.e., mechanically or enzymatically digested, from kidney tissue or isolated from a heterogeneous in vitro culture of mammalian kidney cells.

[0103] Culturing a heterogeneous mixture of kidney cells under hypoxic culture conditions prior to separation on a density gradient results in an enhanced distribution and composition of cells in both the B4 and B2 fractions, which was unexpectedly discovered. An enrichment of oxygen-dependent cells from B2 to B4 was observed for kidney cells isolated from both diseased and non-diseased kidneys. Without being bound by theory, this may be due to one or more of the following phenomena: 1) selective survival, death, or proliferation of specific cell components during the hypoxic culture period, 2) alteration of cell granularity and / or size in response to hypoxic culture, thereby achieving an alteration in buoyant density and subsequent localization during density gradient separation, 3) alteration in gene / protein expression of cells in response to the hypoxic culture period, thereby resulting in the differentiation characteristics of the cells within any given fraction of the gradient. Thus, in one embodiment, a cell population enriched for tubular cells, e.g., B2, is hypoxia-tolerant. Without being bound by theory, this may be due to one or more of the following phenomena: 1) selective survival, death, or proliferation of specific cell components during the hypoxic culture period, 2) alteration of cell granularity and / or size in response to hypoxic culture, thereby achieving an alteration in buoyant density and subsequent localization during density gradient separation, 3) alteration in gene / protein expression of cells in response to the hypoxic culture period, thereby resulting in the differentiation characteristics of the cells within any given fraction of the gradient. Thus, in one embodiment, a cell population enriched for tubular cells, e.g., B2, is hypoxia-tolerant.

[0104] Exemplary techniques for separating and isolating the cell populations of the present invention include separation on a density gradient based on the different specific gravities of the different cell types contained within the relevant population. The specific gravity of any given cell type can be affected by the degree of granularity within the cell, the cellular water volume, and other factors. In one aspect, the present invention includes, but is not limited to, humans, dogs, and rodents. In one aspect, the present invention includes, but is not limited to, humans, dogs, and rodents, including Over multiple species, the cell preparations of the present invention, e.g., for the isolation of B2 and B4, provide optimal gradient conditions. In a preferred embodiment, the density gradient is derived from a heterogeneous population of kidney cells to obtain a novel enriched population of tubule cell fractions, i.e., the B2 cell population. In one embodiment, the density gradient is used to obtain a novel enriched population of EPO-producing cell fractions, i.e., the B4 cell population, derived from a heterogeneous population of kidney cells. In other embodiments, the density gradient is used to obtain enriched subpopulations of kidney tubule cells, glomerular cells, and endothelial cells. In one embodiment, both EPO-producing cells and tubule cells are separated from red blood cells and cell debris. In one embodiment, EPO-producing cells, glomerular cells, and vascular cells are separated from other cell types as well as red blood cells and cell debris,

[0105] and subpopulations of tubule cells and collecting duct cells are simultaneously separated from other cell types as well as red blood cells and cell debris. The present invention generated novel cell populations by using in part OPTIPREP® (Axis-Shield) density gradient medium containing 60% non-ionic iodinated compound iodixanol in water. However, one of ordinary skill in the art will understand that any density gradient or other means having the necessary features for isolating the cell populations of the present invention, e.g., immunological It will also be understood by those skilled in the art that it can be used to separate cell subpopulations by sorting should be (forward scatter = reflection of size by flow cytometry, and side scatter = particle reflection of degree). Importantly, the density gradient medium should have low toxicity to the specific cells in question It is that. While the density gradient medium should have low toxicity to the specific cells in question On the other hand, the present invention contemplates the use of a gradient medium that plays a role in the selection process of the cells in question Although not bound by theory, significant cell loss is seen between the loading step and the recovery step, and exposure to iodixanol under gradient conditions suggests that it causes the removal of specific cells, so the cell population of the present invention recovered by a gradient containing iodixanol is considered to be iodixanol resistant. Cells that appear in a specific band after an iodixanol gradient are resistant to any adverse effects of exposure to iodixanol and / or the density gradient. Thus, the present invention also contemplates the use of additional contrast agents that are mild to moderate nephrotoxins in the isolation and / or selection of the cell population of the present invention Furthermore, the density gradient medium should not bind to proteins in human plasma, or else it will have an adverse effect on the important functions of the cells in question. In another aspect, the present invention provides a method for three-dimensional culturing of a renal cell population. In one aspect the present invention provides a method for culturing a cell population by continuous perfusion. In one embodiment the cell population cultured by three-dimensional culture and continuous perfusion shows higher cell confluence and interconnectivity when compared to a cell population cultured statically In another embodiment it does not, it will have an adverse effect on the important functions of the cells in question.

[0106] In another aspect, the present invention provides a method for three-dimensionally culturing a renal cell population. In one aspect the present invention provides a method for culturing a cell population by continuous perfusion. In one embodiment the cell population cultured by three-dimensional culture and continuous perfusion shows higher cell confluence and interconnectivity when compared to a cell population cultured statically In another embodiment In this embodiment, cell populations cultured by three-dimensional culture and continuous perfusion are Higher expression of EPO, as well as cadherin, in the kidney when compared to static cultures of the group. 4 shows enhanced expression of renal tubule-related genes.

[0107] In yet another embodiment, the cell population cultured by continuous perfusion is a cell population cultured in a statically cultured Higher levels of glucose and glutamine consumption when compared to control cell populations. Shows.

[0108] Those skilled in the art will appreciate that other isolation and culture methods known in the art may be used in conjunction with the methods described herein. It will be understood that the described cells may be used.

[0109] Biomaterials (polymer matrices or scaffolds) Bertram et al., U.S. Patent Application Publication No. 20070276507, which is incorporated herein by reference. ), which is incorporated herein in its entirety. can be designed to satisfy any number of overall system, geometric, or spatial constraints. In one embodiment, the matrix of the present invention may be formed into any number of desired configurations. The scaffold may be three-dimensional and adapted to conform to the dimensions and shape of the organ or tissue structure. For example, kidney disease, anemia, EPO deficiency, renal tubular transport deficiency, We report a three-dimensional (3D) matrix study on the use of polymer scaffolds to treat glomerular filtration deficiency. Various different shapes of 3D scaffolds may be used. The polymer matrix is ​​available in different sizes and shapes to accommodate different sized patients. The polymer matrix may also be shaped to accommodate the specific needs of the patient. The sea urchin may be formed in other forms. In another embodiment, the scaffold may be a biocompatible, porous polymer scaffold. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold configuration may range from a hydrogel suspension liquid to a soft, porous scaffold to a porous scaffold that maintains a rigid shape. The scaffold may be a biocompatible, porous polymer scaffold. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold may be an open cell structured polylactic acid (OPLA (registered trademark)), cellulose ether, cellulose, cellulose ester, fluorinated polyethylene, phenols, poly-4-polymethylpentene, polyacrylonitrile, polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate, polycyanoaryl ether, polyester, polyester carbonate, polyether, polyether ether ketone, polyether imide, polyether ketone, polyether sulfone, polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polysulfide, polysulfone, polytetrafluoroethylene, polythioether, polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose, silicone, urea formaldehyde, collagen, laminin, fibronectin, silk, elastin, alginate, hyaluronic acid, agarose, or a polymer or physical blend thereof, but is not limited thereto, and may be formed from various synthetic or naturally occurring materials. The scaffold configuration may range from a hydrogel suspension liquid to a soft, porous scaffold to a porous scaffold that maintains a rigid shape. The scaffold configuration may range from a hydrogel suspension liquid to a soft, porous scaffold to a porous scaffold that maintains a rigid shape.

[0110] The hydrogel may be formed from various polymer materials and is useful in various biomedical applications. The hydrogel is physically described as a three-dimensional network of hydrophilic polymers. The hydrogel may be formed from various polymer materials and is useful in various biomedical applications. The hydrogel is physically described as a three-dimensional network of hydrophilic polymers. can be clarified. Depending on the type of hydrogel, it contains water in various proportions but is insoluble in water as a whole. Despite having a high water content, hydrogels can further bind to a large amount of liquid due to the presence of hydrophilic residues. Hydrogels expand on a large scale without changing their gelatinous structure. The basic physical properties of hydrogels can be specifically modified by the properties of the polymers used and additional special equipment of the product.

[0111] Preferably, the hydrogel is biologically inert and physiologically compatible with mammalian tissues, made of polymers, biologically occurring materials, synthetically derived materials, or combinations thereof. Hydrogel materials preferably do not induce an inflammatory response. Examples of other materials that can be used to form hydrogels include (a) modified alginates, (b) polysaccharides that gel upon exposure to monovalent cations (e.g., gellan gum and carrageenan), (c) polysaccharides that are either very viscous liquids or thixotropic and form gels over time as their structure gradually develops (e.g., hyaluronic acid), and (d) polymer hydrogel precursors (e.g., polyethylene oxide - poly propylene glycol block copolymers and proteins). U.S. Patent No. 6, 224,893 B1 provides details on various polymers suitable for making hydrogels according to the present invention and the chemical properties of such polymers. The properties of the scaffold or biomaterial can enable cells to adhere to and interact with the scaffold or biomaterial and / or provide a porous space in which cells can be trapped.

[0112] is achievable. In one embodiment, the porous scaffold or biomaterial of the present invention is such that one or more cell populations or mixtures are added or deposited on a biomaterial configured as a porous scaffold (e.g., by cell attachment) and / or the pores of the scaffold (e.g., by cell capture). In another embodiment, the scaffold or biomaterial enables or facilitates cell:cell and / or cell:biomaterial interactions within the scaffold to form the structures described herein.

[0113] In one embodiment, the biomaterial used in accordance with the present invention comprises hyaluronic acid (HA) in hydrogel form and contains HA molecules with a size of 5.1 kDa to greater than 2×106 kDa. In another embodiment, the biomaterial used in accordance with the present invention comprises porous foam hyaluronic acid and similarly contains HA molecules with a size of 5.1 kDa to greater than 2×106 kDa. In yet another embodiment, the biomaterial used in accordance with the present invention comprises a polylactic acid (PLA )-based foam having an open porous structure and a pore size of about 50 microns to about 300 microns. In yet another embodiment, a specific cell population (preferably B2 but also B4) provides direct and / or simultaneous synthesis of high molecular weight hyaluronic acid by hyaluronan synthase-2 (HAS-2), particularly after intrarenal transplantation.

[0114] One of ordinary skill in the art will understand that other types of synthetic or naturally occurring materials known in the art may be used to form scaffolds as described herein.

[0115] In one aspect, the present invention is as described herein made from the scaffolds or biomaterials mentioned above. Provide a structure as described.

[0116] Structure In one aspect, the present invention provides a transplantable structure having one or more of the cell populations described herein for treating kidney disease, anemia, or EPO deficiency in a subject in need thereof. In one embodiment, the structure is composed of a biocompatible material or biomaterial, a scaffold or matrix comprising one or more synthetic or naturally occurring biocompatible materials, and one or more cell populations or mixtures of cells described herein deposited or embedded on the surface of the scaffold by attachment and / or capture. In certain embodiments, the structure is composed of a biomaterial and one or more cell populations or mixtures of cells described herein in combination with the biomaterial, coated on the biomaterial component(s), deposited on the biomaterial component(s), deposited in the biomaterial component(s), attached to the biomaterial component(s), captured by the biomaterial component(s), embedded in the biomaterial component(s). In another embodiment, the deposited cell population or cell component of the structure is a first enriched renal cell population enriched for cells that produce oxygen-regulated EPO. In another embodiment, the first renal cell population contains glomerular cells and vascular cells in addition to cells that produce oxygen-regulated EPO. In yet another embodiment, the deposited cell population or cell component(s) of the structure includes both a first enriched renal cell population and a second renal cell population. In some embodiments, the second cell population is not enriched for cells that produce oxygen-regulated EPO. In another embodiment, the deposited cell population or cell component(s) of the structure is a first enriched renal cell population enriched for cells that produce oxygen-regulated EPO. In another embodiment, the first renal cell population contains glomerular cells and vascular cells in addition to cells that produce oxygen-regulated EPO. In yet another embodiment, the deposited cell population or cell component(s) of the structure includes both a first enriched renal cell population and a second renal cell population. In some embodiments, the second cell population is not enriched for cells that produce oxygen-regulated EPO. In yet another embodiment, the deposited cell population or cell component(s) of the structure includes both a first enriched renal cell population and a second renal cell population. In some embodiments, the second cell population is not enriched for cells that produce oxygen-regulated EPO. In this case, the second cell population is concentrated with respect to the renal tubular cells of the kidney. In another embodiment the second cell population is concentrated with respect to the renal tubular cells of the kidney and contains collecting duct epithelial cells. In other embodiments, the renal tubular cells of the kidney are megalin, cubilin, hyaluronic acid synthase 2 (HAS2), vitamin D3 25-hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad), aquaporin-1 (Aqp 1), aquaporin-2 (Aqp2), RAB17, member-RAS oncogene family (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containing ion transport regulator-4 (Fxyd4), solute carrier family 9 (sodium / hydrogen exchanger), member-4 (Slc9a4), aldehyde dehydrogenase 3 family, member- B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member-A3 (Aldh 1a3), and calpain-8 (Capn8), including but not limited to, the expression of one or more renal tubular cell markers.

[0117] In one embodiment, the cell population deposited or combined on the biomaterial or scaffold to form the structure of the present invention is derived from various sources such as autologous, allogeneic, or syngeneic (autologous or syngeneic transplantation) sources. One of ordinary skill in the art will understand that there are several suitable methods for depositing the cell population on the biomaterial or otherwise combining to form the structure.

[0118] One aspect, the structure of the present invention is suitable for use in the methods of use described herein. One of ordinary skill in the art will understand that there are several suitable methods for depositing the cell population on the biomaterial or otherwise combining to form the structure.

[0119] In one aspect, the structure of the present invention is suitable for use in the methods of use described herein. is suitable. In one embodiment, the construct is suitable for administration to a subject in need of treatment of kidney disease, anemia, or EPO deficiency of any cause. In other embodiments, the construct is suitable for administration to a subject in need of improvement or restoration of erythrocyte homeostasis. In another embodiment, the construct is suitable for administration to a subject in need of improvement of renal function.

[0120] Method of Use In one aspect, the present invention provides a method for treating kidney disease, anemia, or EP deficiency in a subject in need thereof using the renal cell populations and mixtures of renal cells described herein. In one embodiment, the present invention comprises administering to a subject a composition comprising a first renal cell population enriched for cells that produce EPO. In another embodiment, the first cell population is enriched for cells that produce EPO, glomerular cells, and vascular cells. In another embodiment, the composition may further comprise one or more additional renal cell populations. In one embodiment, the additional cell population is a second cell population that is not enriched for cells that produce EPO. In another embodiment, the additional cell population is a second cell population that is not enriched for cells that produce EPO, glomerular cells, or vascular cells. In another embodiment, the composition comprises a renal cell population or mixture of renal cells deposited in, deposited on, embedded in, coated with, or entrapped in a biomaterial component(s) to form a transplantable construct as described herein for the treatment of a disease or disorder described herein. In one embodiment, embodiment, the composition comprises a renal cell population or mixture of renal cells deposited in, deposited on, embedded in, coated with, or entrapped in a biomaterial component(s) to form a transplantable construct as described herein for the treatment of a disease or disorder described herein. In one embodiment, In one state, the cell population is used alone to stimulate regeneration in acute or chronic pathological conditions or in combination with other cells or biomaterials such as hydrogels, porous scaffolds, or natural or synthetic peptides or proteins.

[0121] In another aspect, effective treatment of kidney disease, anemia, or EPO deficiency in a subject using the methods of the invention can be observed by various indicators of erythropoiesis and / or kidney function In one embodiment, indicators of erythrocyte homeostasis include, but are not limited to, hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC), reticulocyte count, reticulocyte percentage, mean corpuscular volume (MCV), and red blood cell distribution width (RDW). In certain other embodiments, indicators of kidney function include, but are not limited to, serum albumin, albumin-to-globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus / calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level, and glomerular filtration rate (GFR). Additionally, some indicators of relative health and well-being include, but are not limited to, weight gain or loss, survival rate, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance In one embodiment, the indicators of erythrocyte homeostasis are hematocrit (HCT), hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC), reticulocyte count, reticulocyte percentage, mean corpuscular volume (MCV), and red blood cell distribution width (RDW). In certain other embodiments, the indicators of kidney function are serum albumin, albumin-to-globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus / calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level, and glomerular filtration rate (GFR). Additionally, some indicators of relative health and well-being are weight gain or loss, survival rate, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance hemoglobin (HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC), reticulocyte count, reticulocyte percentage, mean corpuscular volume (MCV), and red blood cell distribution width (RD W). In certain other embodiments, the indicators of kidney function are serum albumin, albumin-to-globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus / calcium ratio, serum potassium, proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level, and glomerular filtration rate (GFR). Additionally, some indicators of relative health and well-being are weight gain or loss, survival rate, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance serum albumin, albumin-to-globulin ratio (A / G ratio), serum phosphorus, serum sodium, kidney size (measurable by ultrasound), serum calcium, phosphorus / calcium ratio, serum potassium proteinuria, urinary creatinine, serum creatinine, blood urea nitrogen (BUN), cholesterol level, triglyceride level, and glomerular filtration rate (GFR). Additionally, some indicators of relative health and well-being are weight gain or loss, survival rate, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance In another embodiment, effective treatment is manifested by stabilization of one or more indicators of kidney function. Stabilization of kidney function is compared to the same in a subject not being treated using the methods of the invention survival rate, blood pressure (mean systemic blood pressure, diastolic blood pressure, or systolic blood pressure), and physical endurance In another embodiment, effective treatment is manifested by stabilization of one or more indicators of kidney function. Stabilization of kidney function is compared to the same in a subject not being treated using the methods of the invention

[0122] In another embodiment, effective treatment is manifested by stabilization of one or more indicators of kidney function In another embodiment, effective treatment is manifested by stabilization of one or more indicators of kidney function. Stabilization of kidney function is compared to the same in a subject not being treated using the methods of the invention Observation of changes in an indicator in a subject treated using the method of the invention when compared to the indicator is revealed. Alternatively, stabilization of renal function is determined by observation of changes in an indicator in a subject treated using the method of the invention when compared to the same indicator in the same subject prior to treatment . Changes in the first indicator can be an increase or decrease in value. In one embodiment, the treatment provided by the invention may include stabilization of the blood urea nitrogen (BUN ) level in the subject, and the BUN level observed in the subject is lower compared to a subject having a similar condition who has not been treated using the method of the invention . In certain other embodiments, the treatment may include stabilization of the serum creatinine level in the subject, and the serum creatinine level observed in the subject is lower compared to a subject having a similar condition who has not been treated using the method of the invention . In another embodiment, the treatment may include stabilization of the hematocrit (HCT) level in the subject, and the HCT level observed in the subject is higher compared to a subject having a similar condition who has not been treated using the method of the invention . In another embodiment, the treatment may include stabilization of the red blood cell (RBC) level in the subject, and the RBC level observed in the subject is higher compared to a subject having a similar condition who has not been treated using the method of the invention . One of ordinary skill in the art will understand that one or more additional indicators described herein or known in the art may be measured to determine effective treatment of kidney disease in a subject . In another aspect, the invention relates to a method of providing erythrocyte homeostasis to a subject in need thereof . . . . . . . .

[0123] . . In one embodiment, the method comprises: (a) administering to a subject a renal cell population as described herein, such as , B2 or B4, or a mixture of renal cells, such as B2 / B4 and / or B2 / B3; and (b) determining in a biological sample from the subject that the level of an erythropoiesis indicator is different as compared to the indicator level of a control, wherein the difference in the indicator level indicates (i) that the subject is responsive to step (a) of the administration, or (ii) erythrocyte homeostasis in the subject. In another embodiment, the method comprises: (a) administering to a subject a composition comprising a renal cell population or a mixture of renal cells as described herein ; and (b) determining in a biological sample from the subject that the level of an erythropoiesis indicator is different as compared to the indicator level of a control, wherein the difference in the indicator level indicates (i) that the subject is responsive to step (a) of the administration, or (ii) erythrocyte homeostasis in the subject. In another embodiment, the method comprises: (a) providing a scaffold of a biomaterial or a biocompatible polymer; (b) depositing, in a manner described herein, a renal cell population or a mixture of renal cells of the invention on or in the biomaterial or scaffold to form a transplantable structure; (c) transplanting the structure into a subject; and (d) determining in a biological sample from the subject that the level of an erythropoiesis indicator is different as compared to the indicator level of a control, wherein the difference in the indicator level indicates (i) that the subject is responsive to step (a) of the administration, or (ii) erythrocyte homeostasis in the subject.

[0124] In another aspect, the present invention relates to a method for providing to a subject in need of both stabilization of renal function and restoration of erythrocyte homeostasis, said subject having both a renal function disorder and anemia and / or EPO deficiency. In one embodiment, the method comprises administering a renal cell population or mixture of renal cells as described herein containing at least one of the following cell types: cells derived from the renal tubule, cells derived from the glomerulus, cells derived from the interstitium, cells derived from the collecting duct, cells derived from stromal tissue, or cells derived from the vasculature. In another embodiment, the population or mixture contains both cells that produce EPO and renal tubular epithelial cells, and the tubular cells are identified by at least one of the following markers: megalin, cubilin, hyaluronan synthase 2 (HAS2), vitamin D3 25- hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin ( Ecad), aquaporin-1 (Aqp1), aquaporin-2 (Aqp2), RAB1 7, member-RAS oncogene family (Rab17), GATA binding protein 3 (G ata3), ion transport regulator 4 containing the FXYD domain (Fxyd4), solute transporter family 9 (sodium / hydrogen exchanger), member-4 (Slc9a4), alde hyde dehydrogenase 3 family, member-B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member-A3 (Aldh1a3), and calpain-8 (Capn8). In this embodiment, treatment of the subject is indicated by improvement of at least one indicator of renal function and improvement of at least one indicator of erythropoiesis, compared to either an untreated subject or the subject's pre-treatment metrics.

[0125] In one embodiment, the present invention provides a method for the preparation of ... containing a renal cell population enriched for EPO-producing cells By administering a mixture of kidney cells, (i) renal disease, anemia, or EPO deficiency can be improved. (ii) a method for stabilizing renal function; and (iii) a method for restoring red blood cell homeostasis. or (iv) any combination thereof, wherein the beneficial effect of administering is The effect of administering a cell population that is not enriched for O-producing cells is greater than that of administering a cell population that is not enriched for O-producing cells. In morphology, the enriched cell population provides serum blood urea nitrogen (BUN). In embodiments, the enriched cell population provides improved protein retention in serum. In an embodiment, the enriched cell population has improved levels of serum cholesterol and and / or triglyceride levels. The population provides improved levels of vitamin D. In one embodiment, the enriched cells The enriched cell population provides an improved phosphorus:calcium ratio compared to non-enriched cell populations. In another embodiment, the enriched cell population has an improved ability to differentiate compared to a non-enriched cell population. In a further embodiment, the enriched cell population results in improved levels of serum creatine compared to non-enriched cell populations. In yet another embodiment, the enriched cell population has an improved ability to differentiate compared to a non-enriched cell population. In a further embodiment, the concentrated cells are The cell populations produced improved levels of red blood cell (RBC) counts compared to non-enriched cell populations. results. In one embodiment, an improved level of hematocrit is restored to 95% of normal healthy levels. In further embodiments, the concentrated cell population results in an improved reticulocyte count as compared to a non-concentrated cell population. In other embodiments, the concentrated cell population results in an improved percentage of reticulocytes as compared to a non-concentrated cell population. In still further embodiments, the concentrated cell population results in an improved level of red blood cell distribution width (RDW) as compared to a non-concentrated cell population. In yet another embodiment, the concentrated cell population results in an improved level of hemoglobin as compared to a non-concentrated cell population. In still another embodiment, the concentrated cell population results in a erythroid hematopoietic response in the bone marrow such that the cellularity of the bone marrow is nearly normal and the bone marrow:erythroid ratio is nearly normal.

[0126] In another aspect, the present invention provides a method of (i) treating kidney disease, anemia, or EPO deficiency, (ii) stabilizing kidney function, (iii) restoring erythropoiesis, or (iv) any combination thereof, by administering a concentrated cell population, and the beneficial effects of administration of the kidney cell population or mixture of kidney cell populations described herein are characterized by an improvement in erythropoiesis when compared to the beneficial effects provided by administration of recombinant EPO (rEPO). In one embodiment, the population or mixture, when administered to a subject in need, results in an improvement in erythropoiesis (as determined by hematocrit, hemoglobin, or RBC count) when compared to administration of recombinant EPO protein. In one embodiment, the population or mixture ​ 、 when administered, provides a level of hematocrit, RBC, or hemoglobin that is about 10% or less lower or higher than that of the control hematocrit, compared to recombinant EPO. In a further embodiment, a single dose or delivery of a population or mixture, when administered, results in an improvement in erythrocyte homeostasis (determined by an increase in hematocrit, hemoglobin, or RBC count) in a treated subject for a period significantly exceeding the period during which a single dose or delivery of recombinant EPO protein results in an improvement in erythrocyte homeostasis. In another embodiment, a population or mixture, when administered at the dosages described herein, does not result in a hematocrit, hemoglobin, or RBC count that exceeds about 110% of the normal level of the corresponding healthy control. In a further embodiment, a population or mixture, when administered at the dosages described herein, provides superior erythrocyte homeostasis (determined by hematocrit, hemoglobin, or RBC count) compared to recombinant EPO protein delivered at the dosages described herein. In another embodiment, recombinant EPO is delivered at a dosage of about 100 IU / kg, about 200 IU / kg, about 300 IU / kg, about 400 IU / kg, or about 500 IU / kg. One of ordinary skill in the art will understand that other dosages of recombinant EPO known in the art may be suitable. Another embodiment of the invention is for the preparation of a drug useful in the treatment of kidney disease, anemia, or EPO deficiency in a subject in need thereof, providing erythrocyte homeostasis to a subject in need thereof, or improving renal function in a subject in need thereof, of at least one of the herein-described

[0127] ​​​​​​​​​​​It is directed to the use of a cell population or a transplantable construct as described herein.

[0128] Another embodiment of the invention is the selection of a specific subpopulation of cells based on specific verified therapeutic properties for the treatment of a kidney disease of a specific cause, based on a specific enriched cell population(s) (as described herein).

[0129] Method and route of administration The cell preparation of the present invention can be administered alone or in combination with other bioactive components.

[0130] The therapeutically effective amount of the renal cell population or mixture of renal cell populations described herein ranges from the maximum number of cells that can be safely tolerated by a subject to the minimum number of cells necessary for the treatment of a kidney disease, e.g., stabilization of one or more renal functions, reduction of the rate of decline, or improvement. In certain embodiments, the methods of the invention are about 10,000 cells / kg, about 20,000 cells / kg, about 30,000 cells / kg, about 40,000 cells / kg, about 50,000 cells / kg, about 100,000 cells / kg, about 200,000 cells / kg, about 300,000 cells / kg about 400,000 cells / kg, about 500,000 cells / kg, about 600,000 cells / kg, about 700,000 cells / kg, about 800,000 cells / kg, about 900,000 cells / kg, about 1.1x106 cells / kg, about 1.2x106 cells / kg, about 1.3x10 6 cells / kg, about 1.4x106 cells / kg, about 1.5x106 cells / kg, about 1.6x 106 cells / kg, about 1.7x106 cells / kg, about 1.8x106 cells / kg, about 1. 9x106 cells / kg, about 2.1x106 cells / kg, about 2.1x106 cells / kg, about 1.2 x 10^6 cells / kg, approximately 2.3 x 10^6 cells / kg, approximately 2.4 x 10^6 cells / kg , approximately 2.5 x 10^6 cells / kg, approximately 2.6 x 10^6 cells / kg, approximately 2.7 x 10^6 cells / kg, approximately 2.8 x 10^6 cells / kg, approximately 2.9 x 10^6 cells / kg, approximately 3 x 10^6 cells / kg, approximately 3.1 x 10^6 cells / kg, approximately 3.2 x 10^6 cells / kg, approximately 3.3 x 10^6 cells / kg, approximately 3.4 x 10^6 cells / kg, approximately 3.5 x 10^6 cells / kg, approximately 3.6 x 10^ 6 cells / kg, approximately 3.7 x 10^6 cells / kg, approximately 3.8 x 10^6 cells / kg, approximately 3.9 x 10^6 cells / kg, approximately 4 x 10^6 cells / kg, approximately 4.1 x 10^6 cells / kg, approximately 4.2 x 10^6 cells / kg, approximately 4.3 x 10^6 cells / kg, approximately 4.4 x 10^6 cells / kg, approximately 4. 5 x 10^6 cells / kg, approximately 4.6 x 10^6 cells / kg, approximately 4.7 x 10^6 cells / kg, approximately 4.8 x 10^6 cells / kg, approximately 4.9 x 10^6 cells / kg, or approximately 5 x 10^6 cells / k g, provides administration of the renal cell population or mixture of renal cell populations described herein . In another embodiment, the dosage of cells administered to a subject may be a single dosage, or a single dosage and additional dosages. In other embodiments, the dosage may be provided by the constructs described herein . In other embodiments, the dosage of cells administered to a subject may be calculated based on the estimated kidney mass or the functional kidney mass.

[0131] A therapeutically effective amount of the renal cell population or mixture thereof described herein can be suspended in a pharmaceutically acceptable carrier or excipient. Such carriers include basal medium supplemented with 1% serum albumin , physiological saline, buffered physiological saline, glucose, water, collagen, al Salts, hyaluronic acid, fibrin glue, polyethylene glycol, polyvinyl alcohol, carboxymethyl cellulose, and combinations thereof, but not limited thereto. The dosage form should be compatible with the mode of administration. Accordingly, the present invention provides the use of a renal cell population or a mixture thereof (e.g., a B2 cell population alone, or mixed with a B3 and / or B4 cell population) for manufacturing a drug for treating renal diseases in a subject. In some embodiments, the drug further comprises a recombinant polypeptide such as a growth factor, chemokine, or cytokine. In a further embodiment, the drug comprises a cell population derived from the human kidney. The cells used for manufacturing the drug can be isolated, induced, or concentrated using any of the variants provided by the methods described herein. not limited to these. The dosage form should be compatible with the mode of administration. Thus, the present invention provides the use of a renal cell population or a mixture thereof (e.g., a B2 cell population alone, or mixed with a B3 and / or B4 cell population) for manufacturing a drug for treating renal diseases in a subject. In some embodiments, the drug further comprises a recombinant polypeptide such as a growth factor, chemokine, or cytokine. In a further embodiment, the drug comprises a cell population derived from the human kidney. The cells used for manufacturing the drug can be isolated, induced, or concentrated using any of the variants provided by the methods described herein. renal cell population or a mixture thereof (e.g., a B2 cell population alone, or mixed with a B3 and / or B4 cell population) for manufacturing a drug for treating renal diseases in a subject. In some embodiments, the drug further comprises a recombinant polypeptide such as a growth factor, chemokine, or cytokine. In a further embodiment, the drug comprises a cell population derived from the human kidney. The cells used for manufacturing the drug can be isolated, induced, or concentrated using any of the variants provided by the methods described herein. In some embodiments, the drug further comprises a recombinant polypeptide such as a growth factor, chemokine, or cytokine. In a further embodiment, the drug comprises a cell population derived from the human kidney. The cells used for manufacturing the drug can be isolated, induced, or concentrated using any of the variants provided by the methods described herein. In some embodiments, the drug further comprises a recombinant polypeptide such as a growth factor, chemokine, or cytokine. In a further embodiment, the drug comprises a cell population derived from the human kidney. The cells used for manufacturing the drug can be isolated, induced, or concentrated using any of the variants provided by the methods described herein. renal cell population or a mixture thereof (e.g., a B2 cell population alone, or mixed with a B3 and / or B4 cell population) for manufacturing a drug for treating renal diseases in a subject. In some embodiments, the drug further comprises a recombinant polypeptide such as a growth factor, chemokine, or cytokine. In a further embodiment, the drug comprises a cell population derived from the human kidney. The cells used for manufacturing the drug can be isolated, induced, or concentrated using any of the variants provided by the methods described herein. renal cell population or a mixture thereof (e.g., a B2 cell population alone, or mixed with a B3 and / or B4 cell population) for manufacturing a drug for treating renal diseases in a subject. In some embodiments, the drug further comprises a recombinant polypeptide such as a growth factor, chemokine, or cytokine.

[0132] The renal cell preparation(s), or a mixture or composition thereof, is formulated according to routine procedures as a pharmaceutical composition suitable for administration to humans. Usually, for example, compositions for intravenous administration, intraarterial administration, or administration within the renal capsule are sterile isotonic aqueous buffer solutions. Optionally, the composition can also contain a local anesthetic to relieve any pain at the injection site. Generally, the components are supplied separately or together in unit dosage forms, for example, as a frozen concentrate in a sealed container such as an ampoule indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or physiological saline. When the composition is administered by injection, an ampoule of sterile water for injection or physiological saline is provided so that the components can be mixed before administration. renal cell preparation(s), or a mixture or composition thereof, is formulated according to routine procedures as a pharmaceutical composition suitable for administration to humans. Usually, for example, compositions for intravenous administration, intraarterial administration, or administration within the renal capsule are sterile isotonic aqueous buffer solutions. Optionally, the composition can also contain a local anesthetic to relieve any pain at the injection site. Generally, the components are supplied separately or together in unit dosage forms, for example, as a frozen concentrate in a sealed container such as an ampoule indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or physiological saline. When the composition is administered by injection, an ampoule of sterile water for injection or physiological saline is provided so that the components can be mixed before administration. renal cell preparation(s), or a mixture or composition thereof, is formulated according to routine procedures as a pharmaceutical composition suitable for administration to humans. Usually, for example, compositions for intravenous administration, intraarterial administration, or administration within the renal capsule are sterile isotonic aqueous buffer solutions. Optionally, the composition can also contain a local anesthetic to relieve any pain at the injection site. Generally, the components are supplied separately or together in unit dosage forms, for example, as a frozen concentrate in a sealed container such as an ampoule indicating the amount of the active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing pharmaceutical-grade sterile water or physiological saline. When the composition is administered by injection, an ampoule of sterile water for injection or physiological saline is provided so that the components can be mixed before administration. renal cell preparation(s), or a mixture or composition thereof, is formulated according to routine procedures as a pharmaceutical composition suitable for administration to humans. Usually, for example, compositions for intravenous administration, intraarterial administration, or administration within the renal capsule are sterile isotonic aqueous buffer solutions. Optionally, the composition can also contain a local anesthetic to relieve any pain at the injection site. Generally, the components are supplied separately or together in unit dosage forms, for example, as a frozen concentrate in a sealed container such as an ampoule indicating the amount of the active agent. It may be provided.

[0133] Pharmaceutically acceptable carriers are partially determined by the particular composition being administered and by the particular method used to administer the composition. Thus, there are a wide variety of suitable dosage forms of pharmaceutical compositions (see, e.g., Alfonso R Gennaro (ed), Remington: The Science and Practice of Pharmacy, formerly Remington’s Pharmaceutical Sciences 2 0th ed., Lippincott, Williams & Wilkins, 2003, which is incorporated herein by reference in its entirety). Pharmaceutical compositions are usually prepared aseptically and substantially isotonic and in full compliance with all of the manufacturing and quality control regulations (GMP) of the U.S. Food and Drug Administration for pharmaceuticals and pharmaceutical excipients. One aspect of the present invention further provides a pharmaceutical preparation comprising, for example, only a B2 cell preparation or in combination with a B3 and / or B4 cell preparation, the renal cell preparation of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the preparation comprises 104 to 109 cells derived from mammalian kidneys.

[0134] One aspect of the present invention provides a method of providing to a subject in need one or more of the cell populations described herein comprising a mixture. In one embodiment, the source of the cell population(s) can be autologous, allogeneic, syngeneic (autologous or syngeneic transplantation), and any combination thereof. If not an autologous source, the method involves administration of an immunosuppressive agent.

[0135] It may contain. Suitable immunosuppressive drugs include azathioprine, cyclophosphamide, mizoli bine, cyclosporine, tacrolimus hydrate, chlorambucil, lobenzarit disodium um, olaranolfin, alprostadil, gusperimus hydrochloride, biosinsolve, mu romonab, alefacept, pentostatin, daclizumab, sirolimus, mycopheno- late mofetil, leflunomide, basiliximab, dornase alfa, bin darid (bind arid), cladribine, pimecrolimus, ilodecakin, certolizumab, efalizu mab, everolimus, anisperimus, galiximab, faralimomab, clofarabine 、rapamycin, cypressumab, Chai-Ling Decoction, LDP-03, CD4, SR-43551, S K&F-106615, IDEC-114, IDEC-131, FTY-720, TSK -204, LF-080299, A-86281, A-802715, GVH-313, HMR-1279, ZD-7349, IPL-423323, CBP-1011, MT- 1345, CNI-1493, CBP-2011, J-695, LJP-920, L-7 32531, ABX-RB2, AP-1903, IDPS, BMS-205820, BM S-224818, CTLA4-1g, ER-49890, ER-38925, ISAt x-247, RDP-58, PNU-156804, LJP-1082, TMC-95A 、TV-4710, PTR-262-MG, and AGI-1096, but not limited to this (see U.S. Patent No. 7,563,822). Those skilled in the art will recognize other suitable immuno suppressive drugs.

[0136] The treatment method of the present invention involves delivering an isolated population of renal cells or a mixture thereof to an individual. It includes. In one embodiment, it is preferable to directly administer cells to the site intended for the benefit. In one embodiment, the cell preparation or its mixture of the present invention is individual in a delivery vehicle delivered to an individual.

[0137] Various means for administering cells to a subject will be apparent to those skilled in the art upon considering this specification. Such methods include injecting cells into the target site of the subject. The cells can be inserted into a delivery device or vehicle that promotes introduction by injection or transplantation into the subject. In certain embodiments, the delivery vehicle can include natural materials. In other specific embodiments, the delivery vehicle can include synthetic materials. In one embodiment, the delivery vehicle mimics the structure of an organ or provides a structure that fits appropriately within the structure. In other embodiments, the delivery vehicle has liquid-like properties. Such a delivery device can include a tube such as a catheter for injecting cells and liquid into the body of the receiving subject. In a preferred embodiment, the tube further has a needle, such as a syringe, that can introduce the cells of the present invention to the desired position of the subject. In some embodiments, a cell population derived from the kidney of a mammal is formulated for administration intravascularly through a catheter (the term "catheter" is intended to include any of various tubular systems for delivering substances to blood vessels). Alternatively, the cells may be inserted into or onto a biomaterial or scaffold including, but not limited to, fibers such as woven fabrics, knitted fabrics, fabrics, meshes, and non-woven fabrics, perforated films, sponges and foams, and beads such as microparticles, nanoparticles. The cells can be prepared in various different forms for delivery. ​ can be achieved. For example, the cells may be suspended in a solution or a gel. The cells are the cells of the present invention may be mixed with a pharmaceutically acceptable carrier or diluent that maintains the viable state. Pharmaceutically acceptable carriers and diluents include physiological saline, buffered aqueous solutions, solvents, and / or include a dispersion medium. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid, and often isotonic. Preferably, the solution is stable under the manufacturing and storage conditions, for example, by the use of parabens, chlorobutanol, pheno -l, ascorbic acid, thimerosal, etc., protected against the contamination of microorganisms such as bacteria and molds. Those skilled in the art will understand that the delivery vehicles used in the delivery of the cell populations and mixtures thereof of the present invention can include a combination of the above characteristics will understand that. will be.

[0138] For example, the administration modes of an isolated renal cell population(s) alone or mixed with B4 and / or B3 of the B2 cell population include systemic, intrarenal (e.g., parenchymal), intravenous, or intra-arterial injection, and direct injection into the tissue of the intended active site, but are not limited to this. Additional administration forms used in accordance with the present invention include direct laparotomy, direct laparoscopy by, transabdominal, or percutaneous, single or multiple injections(s). Further additional administration forms used in accordance with the present invention include, for example, retrograde injection and pyeloureteral injection. by, transabdominal, or percutaneous, single or multiple injections(s). Further additional administration forms used in accordance with the present invention include, for example, retrograde injection and pyeloureteral injection. by, transabdominal, or percutaneous, single or multiple injections(s). Further additional administration forms used in accordance with the present invention include, for example, retrograde injection and pyeloureteral injection. Surgical means of administration include partial nephrectomy and structural transplantation, nephrectomy, partial pyelectomy (par tial pyelectomy), angiogenesis using omentum ± peritoneum, biopsy needle tracking of multiple lesions, a one-step procedure including replacement from conical or pyramidal to cylindrical, and renal pole-like, and, for example, a two-step procedure including an organoid-in vivo bioreactor for re-transplantation, etc. including, but not limited to this. In one embodiment, the cell mixture is delivered simultaneously through the same pathway In another embodiment, each of the cell compositions containing the controlled mixture is controlled simultaneously or temporarily by one or more of the methods described herein and is delivered separately to a specific location or by a specific method in any of the controlled modes of delivery.

[0139] The appropriate dosage of cell transplantation in humans can be determined from existing information regarding cell activity, such as the production of EPO, or can be estimated from dosage tests conducted in preclinical trials The amount of cells can be quantified from in vitro culture and in vivo animal experiments and used to calculate the appropriate dosage of the graft material. Also, to determine whether additional transplantation can be performed or the graft material is reduced accordingly, the patient can be monitored. One or more selected extracellular matrix components of collagen or hyaluronic acid, and / or growth factors, platelet-rich plasma, and one or more other components containing drugs known in the art may be added to the cell population and mixtures thereof of the present invention

[0140] as well. All patents, patent applications, and references listed herein are hereby incorporated by reference in their entirety into this specification. The following examples are presented for illustrative purposes only and are in no way intended to limit the scope of the present invention

[0141] to this specification. in its entirety.

[0142] The following examples are presented for illustrative purposes only and are in no way intended to limit the scope of the present invention It is not intended to be limiting.

Example

[0143] Example 1 - Isolation and Characterization of Bioreactive Renal Cells from Mature Pigs Suffering from Renal Insufficiency For the evaluation of cell composition and characterization by direct comparison with age - matched normal porcine kidney tissue, fresh diseased kidney tissue was provided from a case of spontaneous progressive chronic kidney disease (CKD) with anemia in a group of mature pigs (wild boars). Histological examination of the kidney tissue at the time of collection confirmed a kidney disease characterized by severe diffuse chronic interstitial fibrosis with multiple fibrosis and crescent - forming glomerulonephritis. Clinically,

[0144] hyperazotemia (elevated blood urea nitrogen and serum creatinine) and mild anemia (mild decrease in hematocrit and hemoglobin level) were confirmed by clinical chemistry. Cells were isolated, propagated, and characterized from both diseased and normal kidney tissues.

[0145] Figure 1 shows the fibrosis (blue staining indicated by the arrow) of the diseased kidney tissue revealed by Gomori trichrome staining compared to normal kidney tissue. Cubilin - and megalin - expressing functional tubular cells capable of receptor - mediated albumin transport were propagated from both normal and diseased kidney tissues. Cells expressing erythropoietin (EPO) also existed in the cultures and were maintained through multiple passages and freeze - thaw cycles. Furthermore, molecular analysis confirmed that EPO - expressing cells derived from both normal and diseased tissues responded to in vitro hypoxia conditions using induction of EPO by HIF1α and other hypoxia - regulated gene targets including vEGF.

[0146] ​​​​​​​​​​​ Cells were isolated from porcine kidney tissue by enzymatic digestion using collagenase + dispase, and also isolated in another experiment by performing simple mechanical digestion and explant culture. At passage 2, cell cultures from explants containing cells expressing EPO were exposed to both atmospheric (21% ) and various hypoxic (○ < 5%) culture conditions to determine whether exposure to hypoxia would lead to upregulation of EPO gene expression. As described for rodent cultures (see Example 3), normal pigs showed oxygen-dependent expression and regulation of the EPO gene. Unexpectedly, despite the uremia / anemia state of CKD pigs (hematocrit ○ < 34, creatinine > 9.0), cells expressing EPO were readily isolated and propagated from the tissue, and maintained EPO gene expression under hypoxic control as shown in Figure 2. As shown in Figure 3, the cells in the propagated cultures showed the ability to self-organize into

[0147] tubule-like structures.

[0148] As shown in Figure 4, by observing receptor-mediated uptake of FITC-conjugated albumin by the cultured cells, it was confirmed that functional tubular cells were present in the cultures (passage 3). The green dots (indicated by thin white arrows) represent the uptake of fluorescein-conjugated albumin mediated by megalin and cubilin, receptors specific for tubular cells, suggesting protein reabsorption by functional tubular cells. The blue staining (indicated by thick white arrows) is the Hoechst-stained nucleus.

[0149] Collectively, these data show that even in kidney tissue with severe impairment due to CKD​​ Functional renal tubules and endocrine cells can also be isolated and propagated from porcine kidney tissue as shown. Furthermore, these findings support the development of autologous cell-based therapeutics for the treatment of CKD

[0150] Example 2 - Isolation of Bioreactive EPO Cells from Human Kidneys Cells producing EPO were enzymatically isolated from normal adult human kidneys (as described in Example 1 above) As shown in Figure 5, the isolation procedure resulted in a relative increase in EPO expression after isolation compared to the original tissue. As shown in Figure 6, cells producing human EPO can be maintained in culture while retaining EPO gene expression Human cells were cultured / propagated on untreated tissue culture-treated plastic or plastic coated with a specific extracellular matrix such as fibronectin or collagen type I and it was found that all supported EPO expression over time

[0151] Example 3 - Culture of Cells Expressing Bioreactive EPO and Tubular Cells from Rodent Kidneys Culture In this study, primary renal cells isolated from Lewis rats were used to analyze in vitro EPO expression and tubular marker expression in response to hypoxia and shear stress

[0152] Primary renal cells were isolated from Lewis rats using standard methods applied to mice (Aboushwareb et al., 2008. World J. Urol. Aug ;26(4):295 - 300) and propagated in hypoxic / high oxygen or static / dynamic 3D cultures

[0153] ​​​​​​​The oxygen dependence of cells producing EPO was determined by culturing the cells under "normal oxygen pressure" culture conditions in the atmosphere (an incubator at 37°C equilibrated with 21% O 2, 5% CO2), and then lowering the oxygen pressure to hypoxic culture (2 % O2, an incubator at 37°C equilibrated with 5% CO2) in order to activate the hypoxia-dependent gene transcription of EPO. Finally, by switching to normal oxygen pressure conditions, the gene transcription occurring under hypoxic conditions will be inhibited. To attach the cells, primary kidney cells were cultured on both 2D (2D) plates and 3D (see the example of Cultisphere below) structures under normal oxygen pressure (usually for 48 hours). Then, the attached cells were transferred to a hypoxic incubator and cultured for a period of 4 8 hours. After the last time point of 48 hours of hypoxic culture, the cells were returned to normal oxygen pressure culture. Cells from 3 plate replicates were collected at predetermined time points first under normal oxygen pressure, then under hypoxic culture, and finally returned to normal oxygen pressure culture. The collected samples were snap-frozen in liquid nitrogen and stored at -80°C before analysis. Total mRNA was isolated from each replicate, and gene expression analysis was performed by synthesizing cDNA from total mRNA, and relative gene expression was determined using real-time quantitative PCR (qrtpcr). In addition to 3 plate replicates, two technical replicates were analyzed using qrtpcr, and a total of 6 replicates were obtained for each time point of each culture condition. After the last time point of 48 hours of hypoxic culture, the cells were returned to normal oxygen pressure culture. Cells from 3 plate replicates were collected at predetermined time points first under normal oxygen pressure, then under hypoxic culture, and finally returned to normal oxygen pressure culture. The collected samples were snap-frozen in liquid nitrogen and stored at -80°C before analysis. Total mRNA was isolated from each replicate, and gene expression analysis was performed by synthesizing cDNA from total mRNA, and relative gene expression was determined using real-time quantitative PCR (qrtpcr). In addition to 3 plate replicates, two technical replicates were analyzed using qrtpcr, and a total of 6 replicates were obtained for each time point of each culture condition. A was isolated, and gene expression analysis was performed by synthesizing cDNA from total mRNA, and relative gene expression was determined using real-time quantitative PCR (qrtpcr). In addition to 3 plate replicates, two technical replicates were analyzed using qrtpcr, and a total of 6 replicates were obtained for each time point of each culture condition. In addition to the 3 plate replicates, two technical replicates were analyzed using qrtpcr, and a total of 6 replicates were obtained for each time point of each culture condition. Six replicates in total were obtained for each time point of each culture condition.

[0154] Cultisphere-S gelatin microcarrier beads Primary kidney cells were isolated from young rats using standard methods. Approximately 200,000 cells were placed on sterile Cultisphere-S (Sigma- Aldrich, catalog number M9043) macro-porous gelatin microcarriers beads (130 - 380 μm) were placed in a culture containing 200 μl of a 50% (v / v) slurry. They were placed in either a static condition (the plate containing the cells did not receive any movement during the experiment) or a dynamic condition (constantly moving) in an oxygen chamber with either 2% or 21% oxygen. During the test (for 7 days), samples were periodically collected from each condition. Three samples were collected per day from each condition. The gene expression of all samples was normalized to the starting material, which was an unfractionated cell suspension of primary cells from the initial isolation. QRTPCR was performed to examine the expression of tubules, as well as endocrine cell markers, E-cadherin, and EPO, respectively.

[0155] Results: EPO expression in cultured endocrine cells was upregulated by dynamic culture and / or hypoxia, as opposed to static culture. The results of (+) EPO expression in dynamic culture at 3D with an oxygen level of 21% in the atmosphere are shown in Figure 7. Figure 8 represents (+) EPO expression in dynamic culture at 3D with a low oxygen level (2%). Figure 9 also shows the expression of (+) tubule genes in dynamic culture during long-term culture. Both hypoxia and dynamic 3D culture significantly increased (p < 0.05) EPO expression compared to hyperoxia and static culture, respectively. Figure 10 shows EPO expression in hypoxic culture compared to normoxic culture. Figure 11 shows the stimulation of EPO expression by dynamic 3D culture in vitro. The increased expression of EPO was always accompanied by an increased expression of HIF1α, which is its regulatory factor, and other HIF1α target genes such as VEGF. Therefore, EPO expression in cultured neonatal kidney cells was mediated by HIF1α. ​​​​​​​​​​​It is clear that it is controlled by the oxygen level. The above results show that bioreactive primary rodent kidney cells that retain the control of EPO expression by gene transfer can be isolated and propagated in vitro. be isolated and propagated in vitro.

[0156] Example 4 - 3D Structure and Comparative Cultures To determine the best in vitro adaptation of the in vivo functionality (such as therapeutic potential, etc.) of the new tissue / organ constructs containing cells, scaffolds, and media, a number of three - dimensional culture constructs were designed. The new tissue / organ constructs were designed as follows: Primary kidney cell cultures (containing both EPO - producing cells and kidney tubular cells) were seeded onto porous cylindrical scaffolds with a diameter of 5 mm and a height of 5 mm. The cells were seeded at a density of 500,000 - 1,000,000 cells / scaffold and cultured in a prototype multi - well perfusion system (MPS, BD Technologies), providing continuous unidirectional fluid flow throughout the experiment. The medium contained either DMEM + 10% FBS (medium A) or a 1:1 mixture of DMEM + 10% FBS and KSFM medium (medium B). The scaffolds evaluated in these experiments included open - cell structure polylactic acid (OPLA) and collagen 1 scaffolds (both from BD), as well as polyglycolic acid - based scaffolds (PGA) fabricated using standard methods. Characteristics included: Pore size and structure sufficient to allow liquid to flow through the cell - scaffold complex; Scaffold structure and composition providing a microenvironment that allows cell - scaffold and cell - cell interactions; Expressing and / or producing and / or transporting proteins and / or molecules involved in kidney regeneration and / or homeostasis, or expressing

[0157] ​​​​​​​​​​​​ The presence of cells having the potential to produce and / or transport. For example, a preferred 3D scaffold containing open-cell structured polylactic acid (OPLA) was seeded with mammalian renal cells and subjected to in vitro culture in a bioreactor device that provided continuous perfusion of the culture medium throughout the scaffold. The in vitro conditioned scaffold + cell complex was characterized as follows: The presence of viable, metabolically active cells; cell-cell and cell-material interactions; the expression of renal tubular markers including, but not limited to, megalin, γ-glutamyltransferase (GGT), E -cadherin, and N-cadherin; and the expression of renal endocrine markers including, but not limited to, erythropoietin.

[0158] Results: As described, conditioned media and all protein lysates were recovered from 2D and 3D cultures. ELISA assays were performed to quantify the target proteins in both cell lysates (upper panel of Figure 12) and conditioned media (lower panel of Figure 12).

[0159] After 7 days of perfusion or static culture, the seeded OPLA and Col1 scaffolds were fixed in 10% buffered formalin and paraffin embedded using standard techniques. Hematoxylin -eosin (HE) staining was performed to examine the presence and morphology of the cells. Cell confluency was higher in perfusion culture than in static culture, and cell distribution was more evenly spread throughout the OPLA scaffold compared to the Col1 scaffold (see Figure 13).

[0160] Figure 14 shows scanning electron micrographs of OPLA and Col1 scaffolds after 7 days of (static and perfusion) culture. The results of scanning electron microscope (SEM) images are shown. Perfused cultures showed higher cell confluency and cell organization than static cultures.

[0161] mRNA was isolated from scaffolds or 2D cultures by addition of lysis buffer (Qiagen), and from 3D scaffolds by electrical homogenization (Polytron) in lysis buffer. The purified mRNA subjected to RT-PCR analysis using intron-spanning primers specific to the corresponding target gene showed that 3 / 7 of the 3D constructs examined expressed the target gene (i.e., EPO) over the 5 days of culture. In contrast, no detectable target gene mRNA was seen in the 2D constructs (lanes 8 - 10 in FIG. 15) over 5 days.

[0162] Lane 1 in FIG. 15 represents 3D constructs that reach, over 5 days, an expression level approaching that seen in macroscopically dissected fresh tissue known to express the target gene (lane 21).

[0163] FIG. 16 shows the results (resazurin metabolism) of CellTiter Blue® used to evaluate metabolic activity in scaffolds. The best metabolic responses were obtained from scaffold construct C under both perfused and static conditions, followed by scaffold B and scaffold A, respectively. In all scaffold constructs, perfused cultures were superior to static cultures with respect to resazurin metabolism.

[0164] To examine glucose and glutamine consumption by perfused and static 3D cultures of primary kidney cells, conditioned media were harvested and analyzed on a Nova BioProfile® 400. Glucose consumption was significantly enhanced under perfused conditions compared to static conditions.​​ Glutamine was consumed to some extent under all 3D conditions, and slightly higher consumption was observed under perfusion conditions than under static conditions. The production of glutamate, a byproduct of glutamine metabolism, was higher under perfusion conditions than under static conditions in all scaffold configurations examined, and the same was true for the production of lactate, a byproduct of the glycolytic pathway (see Figure 17).

[0165] Example 5 - Isolation of a heterogeneous population of unfractionated renal cells (Test Substance No. 1) A concentrated population of an unfractionated mixture (UNFX) of renal cells, mainly containing tubular cells but also small subpopulations of collecting ducts, glomeruli, endocrine, vascular, and other cell types, was isolated from whole kidneys as follows:

[0166] Cell donor: Twenty 2-week-old male Lewis rats were sacrificed and their kidneys were harvested. The freshly excised kidneys were placed on 50 mL conical tubes (10 kidneys per tube) containing 50 mL of chilled (4 °C) Hypothermasol (Biolife Solutions, Inc., Bothell, WA) and maintained on ice. The next day, the tubes containing the kidneys were rinsed with 70% ethanol and placed in a biological safety cabinet (BSC) for processing.

[0167] Renal cell isolation procedure: The kidneys were rinsed with 1X PBS (Gibco, Grand Island, NY) containing 50 μg / mL of gentamicin (Sigma, St. Louis, MO), and the connective tissue and renal calyces were manually removed using forceps and scissors. The kidneys were minced finely using sterile forceps and scissors to make a cell / tissue slurry.

[0168] The cell / tissue preparation was enzymatically digested in Krebs buffer containing dispase (4 U / mL) (No. 07193, Stem Cell Technologies, Vancouver, BC) + 5 mM CaCl 2+ and collagenase type IV (300 U / mL) (Worthington, Newark, NJ) on a rocking platform at 37 °C for 30 minutes. Subsequently, the resulting cell suspension was filtered through a cell strainer with 70 μm pores (BD Biosciences, Franklin Lakes NJ) and placed into sterile conical 50 mL polypropylene tubes containing 50:50 renal cell growth medium (1:1 high glucose DMEM: KSFM). Then, the suspension was centrifuged at 300 × g for 5 minutes and resuspended in 10 mL of 50:50 renal cell growth medium. 10 mL of the cell suspension was aliquoted into two 15 conical tubes (5 mL each). 5 mls of 3 0% w / v Optiprep (Sigma, St. Louis, MO) was added to each tube and

[0169] inverted 6 times. After mixing, 1 milliliter of 1X PBS was carefully layered on top of each suspension to form a layer. The tubes were centrifuged at 800 × g for 15 minutes without interruption (room temperature). The cell band (containing viable new renal cell prototype No. 1) was removed using a sterile 10 mL pipette, diluted 5-fold in 5 0:50 renal cell growth medium (made with low glucose DMEM), and centrifuged at 300 × g for 5 minutes. After centrifugation, the supernatant was carefully removed, and the cell pellet was resuspended and counted in 10 mL of 5 0:50 (low glucose) renal cell growth medium. A sample of 500,000 cells was taken for gene expression assays. The total yield of viable cells was 230 × 106 (94% viability) .

[0170] ​​​ A total of 46.8×106 cells (1.17×106 cells per plate) were placed in 40 p100 tissue culture-treated polystyrene plates in 50:50 (low glucose) medium (BD Biosciences, Franklin Lakes NJ) and placed in a standard CO2 incubator (21% O2). After 48 hours, the medium of the cultures was completely exchanged with 50:50 (low glucose) medium and placed in a 2% O2 environment at 37°C. After 24 hours, 2% O2 cells were harvested for transplantation. Each plate was washed once with sterile PBS, subsequently, 5.0 mL of warmed 0.25% Trypsin w / EDTA (Sigma) was added, and it was returned to 37°C for 5 - 7 minutes. Growth medium was added to each plate (5.0 mL), the cell suspension was removed and stored in a sterile conical 50 mL polypropylene tube. Centrifuged at 300 ×g for 5 minutes to pellet the cells, washed twice in sterile PBS, and resuspended in chilled (4°C) PBS and counted using a hemacytometer. Aliquots of 10×106 cells / 100 μL were prepared in chilled sterile PBS. The yield of viable cells after culture was 91×106 (90% viability). The growth multiple (seeding → harvest) was 3.91 - fold.

[0171] Example 6 - Isolation and Substrate Seeding of a Heterogeneous Population of an Unfractionated Mixture of Renal Cells (Test Substance No. 2) Cell donor: Ten 2 - week - old male Lewis rats were sacrificed and their kidneys were harvested. The freshly excised kidneys were placed in a 50 mL round containing 50 mL of chilled (4°C) Hypothermasol (Biolife Solutions, Inc., Bothell, WA) Placed on conical tubes (10 kidneys per tube) and maintained on ice. The next day, the tubes containing the kidneys were rinsed with 70% ethanol and placed inside a biological safety cabinet (BSC) for the process.

[0172] Kidney cell isolation process: The kidneys were rinsed with 1X PBS (Gibco, Grand Island, NY) containing 50 μg / mL gentamicin (Sigma, St. Louis, MO), and the connective tissue was manually removed using forceps and scissors. Sterile forceps and scissors were used to finely mince the kidneys into a cell / tissue slurry.

[0173] The cell / tissue preparation was enzymatically digested at 37 °C for 30 minutes on a rocking platform in Krebs buffer containing dispase (4 U / mL) (No. 07193, Stem Cell Technologies, Vancouver, BC) + 5 mM CaCl 2+ and type IV collagenase (300 U / mL) (Worthington, Newark , NJ). The resulting cell suspension was then filtered through a cell strainer with 70 μm pores (BD Biosciences, Franklin Lakes NJ) and placed into sterile conical 50 mL polypropylene tubes containing 50:50 kidney cell growth medium (1:1 high glucose DMEM:KSFM). The cell suspension was then centrifuged at 300 ×g for 5 minutes and resuspended in 10 mL of 50:50 kidney cell growth medium.

[0174] 10 mL of the cell suspension was aliquoted into two 15 conical tubes (5 mL each). 5 mL of 30 % w / v Optiprep (Sigma, St. Louis, MO) was added to each tube, and 6 ​​​​After mixing, carefully layer 1 ml of 1X PBS on top of each suspension. The tubes were centrifuged at 800 x g for 15 minutes (room temperature) without interruption. The cell band (containing viable neo-renal cell prototype No. 1) was removed by centrifugation and incubated for 50: Dilute 5x with kidney cell growth medium (made with low glucose DMEM) and culture at 300 x g for 5 min. After centrifugation, the supernatant was carefully removed and the cell pellet was transferred to 10 mL of 50:1 suspension. The total viable cell yield was 71 × 10 6 (survival rate 97%).

[0175] Sterilized package of 24 open-cell polylactic acid (Open-Cell Polylactide) Actic Acid:OPLA® scaffold (BD Biosciences, (Franklin Lakes NJ) was pre-moistened with 50:50 (low glucose) medium. 1 × 10 RK42 neo-kidney cells were then added to each scaffold in 50 μL of 50:50 (low glucose) medium. Cell prototype No. 1 was seeded. The scaffolds were acclimated for 2 hours at 37°C / 21% O2 and then , filled MPS (multi-well perfusion system) unit (BD Technology es, RTP NC) and incubated in 50:50 (low glucose) medium with 2% O2 for 1 h. The scaffolds were maintained at flow for 5 days. The medium was changed every 2 days (50% of the volume was changed). The cells are carefully removed from the MPS, rinsed in sterile PBS, and transported to prepare them for transplantation. The mixture was then kept at ambient temperature for approximately 2 hours to allow for the reaction to proceed.

[0176] Example 7 - Transplantation of an Unfractionated Mixture of Renal Cells into a Rat Model of Renal Failure and Anemia A neorenal cell prototype containing a heterogeneous mixture of cells including EPO-producing interstitial fibroblasts. To evaluate the therapeutic potential and safety of No.1 (UNFX), proximal and distal tubule epithelial cells, glomerular cells, and endothelial cells were isolated from rat kidneys as described above, and the ability of the new renal cell prototype No.1 to delay or reverse renal failure and / or anemia was evaluated in surgically nephrectomized rodents. The test design is shown in Table 1 below. Non-limiting success factors include: 1) A significant favorable effect on the HCT and / or RBC counts 2) A significant decrease in serum BUN and / or creatinine 3) Histological evidence of erythroid stimulation 4) Histological evidence of kidney regeneration

[0177] According to the test design, 24 female mature Lewis rats (8 - 10 weeks old) were procured from Charles River Laboratories (Wilmington, MA) and assigned to the test as the recipients shown in Table 1 below. Subsets of each group were monitored for the onset of morbidity by daily health assessments and weekly serological and cytological examinations. All recipient animals reached the anemic / uremic state before being treated with the new kidney prototype. Prior to the start of the test, a two-week consecutive increase in serum creatinine in nephrectomized rats (two-fold the control level) was required. The nephrectomized rats were assigned to one of four groups (see Table 1 below). Rats in Group 1 were administered 10 million new renal cell prototype No.1 (test substance Seed 6 new kidney prototype No. 1 cells and attach the OPLA scaffold that has been cultured for 4 days. The rats in Group 3 were treated by attaching an empty OPLA scaffold to the distal pole of the remaining kidney. The rats in Group 4 were not treated. Age-matched control rats without any manipulation were designated as Group 5, and age-matched control rats that underwent sham nephrectomy but no further manipulation were designated as Group 6. For the evaluation of renal function (creatinine and BUN) and erythropoiesis (HCT, RBC, and nucleated RBC (nRBC)), blood was collected from the tail vein of all animals weekly (500 μL). During the test period, the health status of all animals was observed weekly and their body weights were measured. At the end of the test (day 84), the surviving rats were subjected to a swimming endurance test. At autopsy, the thigh, kidney, liver, spleen, heart, and lung were collected, weighed, and processed by formalin fixation and paraffin embedding (FFPE) for histological examination. A part of the kidney was embedded in OCT medium, frozen, and processed using frozen sections. The FFPE tissues were processed and stained with HE.

[0178]

[0179] Table 1: Test Design TIFF0007685688000001.tif116165

[0180] Time point Day 0, first nephrectomy (50%) Day 7, second partial nephrectomy (removal of 2 / 3 of the remaining kidney) Day 43, first blood collection / serology / hematology day Days 83 - 91, treatment day Day 170, sacrifice / test end day

[0181] Test substance: Test substance No. 1 - A subpopulation of EPO-producing cells isolated as described above in Example 3 New kidney cell prototype No. 1 (UNFX), containing a heterogeneous mixture of cultured kidney cells. Test substance No. 2 - As described above in Example 4, containing a subpopulation of cells that produce EPO New kidney structure prototype No. 1, containing an OPLA® scaffold seeded with a heterogeneous mixture of cultured kidney cells Structure prototype No. 1. Test substance No. 3 - OPLA® scaffold

[0182] Female recipient 5 / 6 nephrectomized Lewis rats: 13 female Lewis rats (8 - 10 weeks old) were subjected to two - stage surgical 5 / 6 nephrectomy as described above at Charles Rivers Laboratories (Wilmington, PA). Briefly, during the first phase of the procedure, a ventral midline incision was made on the abdomen and a sterile drape was applied. The intestine was retracted laterally to expose the right kidney. The kidney was dissected from the surrounding tissue.

[0183] The kidney was looped around at each pole at the 1 / 3 position of the suture thread. The suture thread was gently tied around the kidney. One - third of the kidney was excised just beyond the ligature thread at each end. The abdominal incision was closed with suture thread and wound clips. During the second phase of the procedure, and 1 week after the first step, the animals were anesthetized and prepared as described above. The dorsal part of the lumbar region was shaved. A caudal - cranial skin incision was made on the left side of the animal's spine so that the cranial end was just below the rib cage. The abdomen was entered. The kidney was dissected from the surrounding tissue and gently pulled away from the incision. The adrenal gland, which was loosely attached to the anterior pole of the kidney by connective tissue and fat, was gently dissected by peeling off the attachment and returned to the abdominal cavity. The renal vessels and ureter were cauterized. Then, the vessel immediately distal to the cauterized part was ligated. The kidney was removed from the abdominal cavity. The abdominal incision was closed with suture thread and wound clips. The animals were allowed to recover for 1 week before being used in the experiment. The animals were then randomly divided into three groups of 5 animals each. The kidney was removed by excising the ureter. The incision was closed with sutures and wound clips. Closed.

[0184] As a control, sham nephrectomy was performed on 6 age-matched controls (both procedures also involved surgical anesthesia, laparotomy, and laparotomy closure). Additionally, 5 age-matched untreated subjects were obtained. After recovery, all rats were isolated for 5 days. Each rat was assigned to a group and given a numerical identifier, which was recorded on a cage card along with the test number, vendor, and test supervisor. One rat per cage was housed and fed Purina Certified Regular Rodent Chow No. 5002. Water was provided ad libitum. After recovery, all rats were isolated for 5 days. Each rat was assigned to a group and given a numerical identifier, which was recorded on a cage card along with the test number, vendor, and test supervisor. One rat per cage was housed and fed Purina Certified Regular Rodent Chow No. 5002. Water was provided ad libitum. Clinical parameter survival assessment: Body weight was observed and recorded weekly using a calibrated analytical balance. Clinical parameter survival assessment: Body weight was observed and recorded weekly using a calibrated analytical balance. Clinical parameter survival assessment: Body weight was observed and recorded weekly using a calibrated analytical balance. Clinical parameter survival assessment: Body weight was observed and recorded weekly using a calibrated analytical balance.

[0185] Clinical parameter survival assessment: Body weight was observed and recorded weekly using a calibrated analytical balance. Clinical parameter survival assessment: Body weight was observed and recorded weekly using a calibrated analytical balance.

[0186] Hematology / Clinical chemistry: Starting on day 43, blood and serum were collected weekly from Antech for measurement of hematocrit (HCT), red blood cell count (RBC), nucleated red blood cell count (nRBC), creatinine (CRE), and blood urea nitrogen (BUN). Blood was aseptically collected from the tail or saphenous vein and placed in a blood or serum collection tube (both from BD, Franklin Lakes, NJ). To control for within-day variation, blood was collected between 8:00 AM and 12:00 PM. Hematology / Clinical chemistry: Starting on day 43, blood and serum were collected weekly from Antech for measurement of hematocrit (HCT), red blood cell count (RBC), nucleated red blood cell count (nRBC), creatinine (CRE), and blood urea nitrogen (BUN). Blood was aseptically collected from the tail or saphenous vein and placed in a blood or serum collection tube (both from BD, Franklin Lakes, NJ). To control for within-day variation, blood was collected between 8:00 AM and 12:00 PM. Hematology / Clinical chemistry: Starting on day 43, blood and serum were collected weekly from Antech for measurement of hematocrit (HCT), red blood cell count (RBC), nucleated red blood cell count (nRBC), creatinine (CRE), and blood urea nitrogen (BUN). Blood was aseptically collected from the tail or saphenous vein and placed in a blood or serum collection tube (both from BD, Franklin Lakes, NJ). To control for within-day variation, blood was collected between 8:00 AM and 12:00 PM. Hematology / Clinical chemistry: Starting on day 43, blood and serum were collected weekly from Antech for measurement of hematocrit (HCT), red blood cell count (RBC), nucleated red blood cell count (nRBC), creatinine (CRE), and blood urea nitrogen (BUN). Blood was aseptically collected from the tail or saphenous vein and placed in a blood or serum collection tube (both from BD, Franklin Lakes, NJ). To control for within-day variation, blood was collected between 8:00 AM and 12:00 PM. Hematology / Clinical chemistry: Starting on day 43, blood and serum were collected weekly from Antech for measurement of hematocrit (HCT), red blood cell count (RBC), nucleated red blood cell count (nRBC), creatinine (CRE), and blood urea nitrogen (BUN). Blood was aseptically collected from the tail or saphenous vein and placed in a blood or serum collection tube (both from BD, Franklin Lakes, NJ). To control for within-day variation, blood was collected between 8:00 AM and 12:00 PM. Hematology / Clinical chemistry: Starting on day 43, blood and serum were collected weekly from Antech for measurement of hematocrit (HCT), red blood cell count (RBC), nucleated red blood cell count (nRBC), creatinine (CRE), and blood urea nitrogen (BUN). Blood was aseptically collected from the tail or saphenous vein and placed in a blood or serum collection tube (both from BD, Franklin Lakes, NJ). To control for within-day variation, blood was collected between 8:00 AM and 12:00 PM.

[0187] Surgical procedure: Anesthesia / Sedation / Analgesia: The rats were sedated prior to surgery by first administering 0.05 cc (0.3 mg / mL) of buprenorphine (Buprenex) in a syringe attached to a 26-gauge needle (IP). Then, they were first placed in a chamber with 4 - 5% isoflurane. Anesthesia / Sedation / Analgesia: The rats were sedated prior to surgery by first administering 0.05 cc (0.3 mg / mL) of buprenorphine (Buprenex) in a syringe attached to a 26-gauge needle (IP). Then, they were first placed in a chamber with 4 - 5% isoflurane. Anesthesia / Sedation / Analgesia: The rats were sedated prior to surgery by first administering 0.05 cc (0.3 mg / mL) of buprenorphine (Buprenex) in a syringe attached to a 26-gauge needle (IP). Then, they were first placed in a chamber with 4 - 5% isoflurane. Positioned, and then maintained anesthesia with isoflurane inhalation anesthetic (3%) throughout the procedure via a nose cone By doing so, the rats were anesthetized with isoflurane inhalation anesthesia. After the surgery, the second dose of Buprenex was administered to each rat, and the third dose was given the next day.

[0188] Surgical preparation: After achieving an appropriate level of anesthesia (evaluated by pinching the toes), the animals were placed in the dorsal recumbent position and the right dorsal lateral area was shaved by applying Betadine (3 times) and ethanol (4 times) concentrically using a No. 5 clipper To enable accurate monitoring of respiration, the area was prepared for aseptic surgery using a sterilized transparent adhesive drape.

[0189] Direct injection of new renal cell prototype No. 1 (test substance No. 1, or UNFX): The right dorsal lateral area was incised longitudinally to expose the peritoneal cavity. The remaining right kidney was isolated and partially withdrawn from the peritoneal cavity using sterilized gauze and blunt-ended surgical forceps New renal cell prototype No. 1 (prep RK40, test substance 1) was gently resuspended, filled into one sterilized 1 cc syringe (B D, Franklin Lakes, NJ), and then a 23G needle was attached. 100 μL of the cell suspension (10 million cells ) was gradually delivered into the renal parenchyma through the needle targeting the corticomedullary junction. A new sterilized 23G needle was used for each animal. When the needle was withdrawn a collagen disk (GelFoam, 2 mm × 2 mm ) was placed at the injection site to relieve bleeding. The kidney was returned to the abdominal cavity, and 1 mL of warmed sterilized physiological saline was added to supply moisture. The muscle wall was closed using 4.0 Vicryl suture, and the skin was closed using a wound clip (both items from Ethicon Inc., Somervi ll, NJ). lle, NJ). Oxygen was administered postoperatively (via inhalation / nose cone) and the animal was monitored until it awoke to a state of consciousness. The animal was monitored until it reached a certain state of consciousness after oxygen administration (via inhalation / nose cone) postoperatively.

[0190] Surgical delivery of scaffold-based test substances (No. 2 and No. 3): The right dorsolateral region was incised longitudinally, exposing the peritoneal cavity. The remaining right kidney was isolated and partially withdrawn from the peritoneal cavity using sterile gauze and blunt surgical forceps. The position of the distal pole of the right kidney was confirmed, and adherent abdominal fat was dissected from the area until dark red renal tissue was observed using a non-tapered surgical technique or forceps. The kidney was exposed using a No. 10 scalpel (Sklar Scientific, West Chester, PA), and the renal parenchyma was excised with the scalpel until an area of approximately 4 × 4 mm was exposed and a small amount of bleeding was visible. A single prototype No. 1 of the new kidney structure (test substance No. 2) or the scaffold only (test substance No. 3) was bisected (to provide a large flat adhesion point with the exposed renal parenchyma) and adhered to the excised area of the renal parenchyma with fibrin glue (Ethicon Inc., Somerville, NJ) (such that the excision surface was on top of the excised area). The abdominal fat was folded over the scaffold and further sealed to the renal surface with fibrin glue, providing an auxiliary blood supply to the adhered scaffold. The kidney was then observed for approximately 1 minute for signs of bleeding. If bleeding occurred, it was stopped with fibrin glue, and then the scaffold with the adhered remaining kidney was returned to a neutral position within the abdominal cavity. Subsequently, 1.0 mL of warmed sterile saline was added to the abdominal cavity to assist with hydration. The muscle wall was closed using 4.0 Vicryl sutures, and the skin was closed using wound clips (both items from Ethicon Inc., Somerville, NJ). Oxygen was administered postoperatively (via inhalation / nose cone). ​​​​​​​​​ Through the [device name], the animals were monitored until they woke up and became conscious.

[0191] Post-operative recovery: After the operation, the animals were recovered in an operating room equipped with a cage containing an absorbent pad (without a fixed floor covering) placed on a warming pad. When the rats woke up and started moving, they were returned to individual cages and observed throughout the rest of the day and the next morning.

[0192] Pre-autopsy procedures: Swimming endurance test: One large circular metal tub (140 cm in diameter, 45 cm deep) was filled with water and maintained at 32°C. Rats (one at a time) were gently placed into the water facing the side of the tank and released. Using a stopwatch, the time from release until the rat stopped swimming and could no longer stay afloat on the water surface was measured and recorded. The rats were dried using a cotton towel and allowed to recover on a warming pad before being returned to their cages. Before each test for each animal, the excrement was removed from the pool and the temperature was checked and recorded.

[0193] Final body weight: The animals were transferred to the autopsy room. The final body weight was measured using an analytical balance and recorded in grams.

[0194] Euthanasia: The rats were placed in a CO2 chamber. Death was confirmed by checking for reflexes due to deep pain and eye examination.

[0195] Autopsy procedures: Cardiac puncture / blood collection: The euthanized rats were placed on an autopsy table and blood was collected directly into serum and blood collection tubes. The serum samples were rotated and serum was collected for a complete serum chemistry panel. Blood samples were collected for a hematology panel.

[0196] Tissue collection: The remaining kidney (or the entire right kidney for controls and suspected treatments) was carefully removed , weighed, and bisected longitudinally. One half was placed in 10% buffered formalin and processed for paraffin embedding and hematoxylin and eosin (HE) staining. The other half was placed in a mold containing OCT embedding medium pre-cooled on dry ice and frozen. The frozen kidney half was maintained on dry ice and stored at -80°C until the frozen sections were ready for Y chromosome analysis. At necropsy, the spleen, liver, , heart, and lungs were removed, weighed, and small incisions were made in each, and then placed in 10% buffered formalin for paraffin embedding and HE staining. Finally, the thighs were removed and the associated stromal tissue was removed. The proximal ends were shaved with a scalpel blade to expose the bone marrow and entire thigh, and then placed in 10% buffered formalin prior to paraffin embedding and HE staining procedures.

[0197] Results Survival rate: Most of the nephrectomized rats could not survive until the end of the test period. Rats in groups 1, 5, and 6 survived until sacrifice on day 84. The mean survival time (days) of untreated nephrectomized rats in group 4 was 48.25 ± 29.8 days. Nephrectomized rats administered only scaffolds (group 3) survived for 25 days after treatment. Rats administered the new kidney structure prototype No. 1 (group 2) survived for an average of 57.5 ± 14.3 days, or 9.25 days longer than untreated nephrectomized rats. Rats in group 1 treated with the new kidney cell prototype No. 1 survived throughout the test period and were sacrificed on day 84 (arrow) along with groups 5 and 6 (Figure 18).

[0198] Body weight: Body weight was measured weekly from the start of the test (day 14) to sacrifice (day 170). The body weight data (g) for each group are shown in Figure 19. Rats in group 5 (control) had an average of 5 Increased by 6% (±5.5%), and the rats in Group 6 (sham surgery) increased by an average of 30.3% (±5.3%). All nephrectomized rats (Groups 1 - 4) showed a significantly lower percentage of weight gain over the test period. Group 1 (New kidney cell prototype No. 1) had an average increase of 13.5%, Group 2 (New kidney structure prototype No. 1) had an average increase of 13.3%, Group 3 (Empty scaffold) had an increase of 7.8%, and Group 4 (Untreated ) had an increase of 13.7%. When examining the weight gain between the time of treatment and the time of sacrifice or death, the differences between groups were even more pronounced (Figure 20). During the treatment period (Day 83 - 170), the rats in Groups 5 and 6 had weight increases of 11.9% and 8.8% respectively, while the untreated nephrectomized rats (Group 4) had an average weight loss of 4.8%. Rats administered only the new kidney cell prototype No. 1, structure, or scaffold (Groups 1, 2, and 3) also had weight loss during the treatment period, but not as much as the untreated nephrectomized rats in Group 4.

[0199] Weekly evaluation of renal function (BUN and creatinine): Throughout the test, measurements of both BUN and creatinine were taken weekly from serum starting at Week 4 until sacrifice on Day 170. The average data for individual rats are shown in Tables 2 and 3 below. Figures 21 and 22 show the time - course measurements of BUN and creatinine between groups as weekly averages respectively. The controls and sham - operated animals [BUN] were on average 19.1±1.9 throughout the test. At the time of transplantation (Weeks 7 and 8 on the graph, arrows), serum BUN increased in all nephrectomized rats, and the average [BUN] among nephrectomized rats was 53.3±19.9. Throughout the test period, the untreated nephrectomized rats in Group 4 showed a continuously increasing [BUN], and the pre - death value reached over 100. ​​​​​​​​​​After treatment with the new renal cell prototype No. 1, the rats in Group 1 showed stabilization of serum [BUN] until the 14th week, and [BUN] increased from that point until sacrifice at the 19th week. The rats in Group 2 administered with the new kidney structure body prototype No. 1 showed lower serum [BUN] than Group 4, but did not show stabilization to the same extent as Group 1. The rats in Group 3 administered only with the scaffold rapidly decreased only after the procedure, as indicated by the rapidly rising [BUN] and death at the 10th week.

[0200] The results of serum creatinine in each group showed the same tendency of therapeutic effect as described for [BUN] (Figure 22). During the entire test period (7th week to 19th week), the serum [creatinine] of the control animals in Group 5 and the sham-operated animals in Group 6 was stable at 0.4 ± 0.5. In contrast, the untreated nephrectomized rats in Group 4 started the test with an average [creatinine] of 1.38 ± 0.75 at the 7th week, reached a maximum of 2.7, and were 2.6 ± 0.15 on average at death. The average [creatinine] of the animals seeded with the scaffold was 2.3 (std. 0.92) at death, representing a slight improvement in Group 4. Interestingly, the nephrectomized rats administered with the new renal cell prototype No. 1 (Group 1) maintained stable [creatinine] levels from 0.8 ± 0.0 at the time of cell transplantation (7th week) to 1 .0 ± 0.0 at the 15th week, and only slightly increased to an average of 1.3 ± 0.18 towards the end of the test (16 - 19th week).

[0201] Examination of BUN and creatinine values at the mid - time points (12th week and 13th week) of the test, where the data of each individual rat is presented as a percentage of (control + sham - operated - Groups 5 and 6), provides a means for examining the variation among rats in each group (Figs. 23 and 24). 12 At both the 12th and 13th weeks, 3 / 5 of the rats with the prototype No. 1 of the new kidney structure in Group 2 had significantly lower BUN and creatinine than the untreated nephrectomized rats in Group 4 . Both ( 2 / 2) of the rats treated with the new kidney cells of Group 1 had significantly lower BUN and creatinine than those in Group 2 or 4.

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[0204] Weekly evaluation of erythropoiesis (HCT, RBC, nRBC): All data are shown in Tables 4 and 5 below. From the 7th week (at the time of treatment) to the 19th week (at the end of the test), the average HCT of the control in Group 5 and the sham surgery in Group 6 were 46.9 ± 0.8 and 46.2 ± 1.2, respectively. In contrast, the nephrectomized animals in Group 4 that did not receive treatment showed an average HCT of 40 ± 2.6 at the 7th week, and then rapidly decreased until they were sacrificed due to illness. The average HCT at the time of sacrifice was 35 .6 ± 1.8. The average HCT at the time of death of the animals with the prototype No. 1 of the new kidney structure in Group 2 was 3 8.4 ± 4.3, showing improvement compared to Group 4 but not reaching the HCT of the control in Group 5. Interestingly, the animals in Group 1 administered with the new kidney cell prototype No. 1 showed improvement in HCT from the pre-transplant average ( 7th week) of 42.8 ± 0.2 to equal values in Groups 5 and 6 at the 14th week (48.9 ± 0.4). The HCT values in Group 1 gradually decreased from the 15th to 19th weeks and reached an average of 36.8 ± 7.1 at the time of sacrifice. The weekly average data are shown in Fig. 25. Separate treatment is shown in Fig. 25. Separate treatment ​Variation among rats in the treatment groups was sometimes high, so it was useful to view the HCT data in an additional format to enable evaluation of individual rat data. Figure 26a provides data from all treatment groups at weeks 12 and 13, expressed as a percentage relative to the control (control = average value from all control and sham-operated animals on these days). RBC and nucleated RBC (nRBC) were also measured weekly. Nucleated RBC was not detected throughout the study and thus not graphed. Mean data are shown in Table 5 below. From week 7 (time of treatment) to week 19 (end of study), the mean RBC counts for the control in group 5 and sham operation in group 6 were 8.32 ± 0.2. Nephrectomized rats without treatment had a mean RBC count of 7.58 ± 0.58 at the beginning of week 7, which decreased rapidly until death, with a mean RBC count at death of 6.5 ± 0.5. Animals in group 2 with the new kidney construct prototype No. 1 had a mean RBC count at death of 6.94 ± 0.7. Animals in group 1 administered the new kidney cell prototype No. 1 showed a stable and consistent improvement in RBC count from 6.71 ± 2.53 at week 8 to 8.03 ± 0.32 at week 18. A sharp decrease (6.6 ± 1.15) was seen in the RBC count at week 19 (end of study). Generally, the weekly mean RBC count was tracked with HCT (Figure 27). Similarly, variation among animals in the various treatment groups was usefully depicted by adding graphs at intermediate time points, whereby the RBC counts of individual animals (weeks 12 - 13) are expressed as a percentage relative to the control (Figure 28). (%)

[0205]

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[0208] Hematology and Clinical Chemistry at Slaughter Electrolyte balance: Serum levels of calcium, potassium, chloride, and phosphorus were measured at slaughter (with the exception of animals that were dead and from which blood could not be collected). All data are shown in Tables 7a - 7c below. The results are summarized in Figure 29. The normal ranges for each parameter measured are indicated by the double - dotted lines on the graph (for reference normal ranges, see Gad(ed.), Animal Models in Toxicology, 2nd edition, (2008), Informa Healthcare USA, New York, NY). Renal failure is usually associated with increases in potassium, sodium, and phosphorus, and decreases in calcium and chloride levels. Nephrectomized rats in Group 4 showed an increase in sodium and phosphorus (but not potassium) and a decrease in chloride level compared to the control. The most consistent electrolyte changes in nephrectomized rats were a decrease in chloride level and an increase in phosphorus level. Treatment with the new renal cell prototype No. 1 (Group 1) resulted in a decrease in phosphorus and calcium, and a slight increase in chloride compared to Group 4. (See Figure 30).

[0209] When considering the serum phosphorus levels of the rats analyzed individually, 2 / 4 of the rats had phosphorus levels as low as those of the controls and sham - operated rats in Groups 5 and 6, and the rats treated with the new renal cells in Group 1. It is clear that the response of the rats in Group 2 is heterogeneous, similar to the BUN, creatinine, HCT, and RBC values.

[0210] ​​​​​​​​​​​​​Serum proteins: Except when the rats were dead and blood could not be collected, at the time of slaughter the levels of total serum protein, serum albumin, and serum globulin were measured in all rats. For the control and sham-operated rats in Groups 5 and 6, serum albumin and globulin were within the normal range. The untreated nephrectomized rats in Group 4 had significantly lower serum albumin and total protein compared to the control and sham-operated rats. Treatment with New Kidney Prototype No. 1 (Group 1) resulted in a slight recovery of serum albumin and total serum protein (Figure 31). Treatment with New Kidney Structure Prototype No. 1 (Group 2) also resulted in a slight recovery of total serum protein. The individual rat data for serum albumin and total protein are presented in Figure 32 as percentages relative to the control.

[0211] Liver function: Liver function was evaluated by measuring bilirubin, AST, ALT, GGT, and ALP. All data for these tests are shown in Tables 7a - 7c. The mean serum AST was above the reported normal range in the sham-operated rats in Group 6 and the rats treated with Nx + New Kidney Prototype No. 1 in Group 1 (Figure 33). The control in Group 5 was within the normal range, as were Groups 4 and 2, although the degree of variation was high. With the exception of Group 2 (New Kidney Structure Prototype No. 1 ), all groups showed mean serum ALT and ALP higher than the reported normal range for rats. Bilirubin levels were within the reported normal range, and no differences were seen between the treatment groups.

[0212] Lipids and sugars: Cholesterol, triglyceride, and glucose in the serum recovered from the animals at the time of slaughter were also measured. All data are presented in tabular form in Attachment F. By type of treatment Nevertheless, in the nephrectomy groups (1, 2, and 4), both mean serum cholesterol and tri glyceride levels were significantly elevated. The mean value of triglyceride treated with the new renal cell prototype No. 1 in Group 1 was significantly higher than that of the control and sham operation (Groups 5 and 6), but it should be noted that it was also higher than those of Groups 4 and 2 at the same time (Figure 34). The mean serum glucose level was higher than the normal range reported for all groups. The rats treated with the new renal cells in Group 1 showed serum [glucose] equivalent to that of the control and sham operation, while the rats in Groups 4 and 2 showed slightly lower serum [glucose]. To understand the variation among rats in each group, the individual rat data expressed as a ratio to the control + sham operation are shown in Figure 35.

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[0216] Hematology at sacrifice Hemoglobin: Blood was collected at sacrifice to measure hemoglobin (Hb), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). All data are shown in Tables 7a - 7c. The nephrectomized untreated rats in Group 4 showed significantly lower [Hb] compared to the control and sham operation (Figure 36), and as expected, a decrease in both HCT and RBC count was observed in Group 4. The mean [Hb] in Groups 1 and 2 ​​​​​​​Both were high, and many rats returned to the normal range (see the individual data in Figure 37). . MCH was relatively equal in all rats, and the hemoglobin concentration per RBC suggested similarity among treatments. An increase in [Hb] was observed in rats in Groups 1 and 2 treated with New Kidney Prototype No. 1 or New Kidney Structure Prototype No. 1, which was consistent with the increase in RBC count and hematocrit observed in these rats. Overall, this data suggests erythropoiesis and stimulation of the oxygen-carrying brain in treated rats.

[0217] WBC count and RBC count and composition: Except for cases where rats were dead and blood could not be collected, white blood cell count (WBC) and red blood cell count (RBC) were measured from all rats at the time of sacrifice. All hematological data are presented in Tables 7a - 7c. Furthermore, the total of the WBC population from each rat was evaluated for the relative percentages (%) of lymphocytes, monocytes, basophils, neutrophils, eosinophils, and large unstained cells (LUC). As shown in Figure 38, the mean WBC was within the normal range in all groups except for the untreated nephrectomized rats in Group 4 where WBC decreased. Figure 39 reveals the differences in the composition of WBC among groups. Note the relatively low percentage (%) of lymphocytes and relatively high percentage (%) of neutrophils in the nephrectomized untreated rats in Group 4 administered New Kidney Structure Prototype No. 1 and the nephrectomized rats in Group 2.

[0218] Regarding the red blood cell population, the number and percentage (%) of reticulocytes were measured at the final blood collection (see Figure 40). RBC and HCT have already been reported (Figures 26 - 2 ). ​​​​​​​​(see reference 9). The number and percentage of reticulocytes in untreated nephrectomized rats in group 4 %) were decreased compared to the controls and sham surgeries in groups 5 and 6. In both treatment groups (1 and 2), there was a slight improvement compared to group 4, but the difference was not statistically significant . RDW (measuring the variation in RBC width) and MCV (mean corpuscular volume) were measured, but no significant differences between groups were found (data not shown). All hematological data are shown in Tables 7a - 7c.

[0219] Platelets: Platelet count and mean platelet volume (MPV) were measured in blood collected at sacrifice. The platelet count was slightly above the normal range reported in all groups. No significant differences were found in platelet count or MPV between treatment groups (see Figure 41) .

[0220] Swimming endurance: At the end of the test, just prior to sacrifice on day 84, the remaining rats were subjected to a swimming endurance test as described in the methods section. A swimming test was performed between the new kidney cell - treated rats in group 1 and the controls and sham surgeries (groups 5 and 6). As shown in Figure 42, healthy rats swam for an average of 2 minutes and 12 seconds, with a reasonable degree of variation (±33 seconds) between rats. Also, this test confirmed that the functional

[0221] Histopathology Representative photographs of each examined tissue can be seen in Figures 43 - 46. An overview of the findings for each tissue between groups is provided below.

[0222] Liver: Compared to the controls and sham surgeries in groups 5 and 6, group 4 (Nx) or group 2 (Nx No significant changes were observed in the liver parenchyma and / or portal triads of the (+Neo-kidney constructs). In contrast, local areas of hematopoiesis were seen in the sinusoids of the rats in Group 1 (Nx + Neo-kidney cells).

[0223] Kidney: Compared to Groups 5 and 6, all other test groups showed progressive degeneration of the glomeruli and tubules of the kidney, associated with loss of structure characterized by an ill-defined boundary between the cortex and medulla, cystic spaces, periglomerular fibrosis, replacement of the glomerular tuft by avascular glassy material, hemosiderin pigment deposition, and multifocal tubular regeneration.

[0224] Spleen: No significant histological differences or changes were observed between the controls and sham surgeries in Groups 5 and 6 and Group 1 (Nx + Neo-kidney cell injection). Examination of the subcapsular colored medullary spaces in the animals of Groups 2 and 4 showed moderate and marked decreases, respectively, in adult red blood cells (RBCs) and RBC progenitor cells.

[0225] Bone marrow: Compared to the controls and sham surgeries in Groups 5 and 6, the cellularity of the bone marrow and the bone marrow to erythroid ratio in the rats of Group 1 appeared equal. In contrast, the bone marrow of the animals in Groups 2 and 4 was characterized by moderate and marked decreases, respectively, in cellularity. As shown in Figure 26, delivery of new kidney cells in vivo to uremic / anemic rats stimulated the bone marrow, particularly the erythroid population. Figure 26 further shows that delivery of new kidney cells in vivo to uremic / anemic rats also stimulated overall cellularity, which persisted for at least 3 months after delivery.

[0226] ​​​​​​​​​​​​​​As shown above, the new renal cell prototype No. 1 delivered alone or on a 3D scaffold ( UNFX) had a regenerative effect on erythropoiesis and erythrocyte homeostasis in a surgical 5 / 6 nephrectomy model, as determined by the temporal analysis of RBC count and HCT, and as confirmed by the final blood tests with other parameters (Hb, MHC, reticulocytes). Among animals, there was some variability in the effect, particularly in rats treated with the new kidney structure prototype No. 1 in group 5, which may be due to variations in the retention of the scaffold by the kidney or possibly due to the occurrence of an unknown scaffold collapse within the renal parenchyma. Importantly, in each given rat, the improvement in HCT or creatinine transitioned to an improvement in RBC count and BUN values, resulting in an overall favorable effect, as confirmed by the observation. Figure 47 plots the HCT data against the serum [creatinine] of each rat. All group 5 and 6 controls and sham surgeries grouped in the upper right quadrant, meaning higher HCT and lower creatinine. 2 / 2 and 2 / 5 of the rats in groups 1 and 2 were away from the upper right quadrant with groups 5 and 6. 1 / 3 of the treated rats in group 2 showed an improvement in creatinine compared to group 4, but no improvement in HCT, so it is shown in the upper right quadrant. The remaining two rats in group 2 showed little or no improvement in any parameter and thus grouped in the lower left quadrant (high creatinine, low HCT) with the rats in group 4 . In conclusion, the above results indicate that the transplantation of new renal cells (group 1) improved the survival rate of 5 / 6 nephrectomized rats . . . . . . . .

[0227] . Indicates improvement. The rats in group 1 of 2 / 2 reached the 3-month point after treatment and were sacrificed on day 84 together with the sham operation. Transplantation of new kidney cells (group 1) reversed anemia in this model for approximately 12 weeks, as evidenced by the return of HCT and RBC to the normal range. Furthermore, Figure 47 shows that delivery of new kidney cell prototype No. 1 in vivo to uremic / anemic rats restored hematocrit to normal levels and promoted survival up to and beyond that of untreated uremic / anemic rats. Also, as shown above, transplantation of new kidney cells regulated erythrocyte homeostasis for a 12-week period, as evidenced by the failure of the treatment to overcorrect anemia and cause polycythemia. Interestingly, transplantation of new kidney cells resulted in stabilization of renal function, as evidenced by the stabilization of serum BUN and CREA from treatment through week 12. New kidney structure prototype No. 1 resulted in improvement in both HCT and stabilization of renal function in 2 / 5 of the rats at the midpoint of the study. At the time of death of the rats due to the disease, renal function and erythrocyte function were somewhat better than the values at the time of death of the sham-operated untreated rats in group 4 due to the disease. The observation of heterogeneous properties in the cultured new kidney cells used in these experiments, combined with the obvious therapeutic effect observed after transplantation, facilitated the identification of specific cell component(s) within the population responsible for the therapeutic effect. Also, the above results indicate that when new kidney cell prototype No. 1 (2 / 2) and new kidney structure prototype No. 1 ( 3 / 5) were delivered, clear and significant favorable effects on HCT and RBC counts were observed. The favorable effect of new kidney cell prototype No. 1 was rapid (after treatment or

[0228] and was observed, indicating a clear and significant favorable effect on HCT and RBC counts. The favorable effect of new kidney cell prototype No. 1 was rapid (after treatment or and was for up to 1 week) and persisted up to 3 months after treatment. This study also showed that the new renal cell prototype No. 1 ( 2 / 2) and the new kidney structure prototype No. 1 (3 / 5) brought about stabilization of renal function and delayed disease progression compared to untreated nephrectomized rats. Furthermore, clear histological evidence suggested that the new renal cell prototype No. 1 (2 / 2) provided sufficient stimulation of erythroblasts in the bone marrow to result in a normal cellularity and M:E ratio at the time of sacrifice (3 months after treatment). Moreover, rats with the new kidney structure prototype No. 1 (group 2) did not show normal bone marrow histology at the time of sacrifice, but had better cellularity and the presence of erythroid hematopoietic cells than untreated nephrectomized rats in group 4. Histological evidence obtained from this study also suggested that the new renal cells in group 2 stimulated a mild histological improvement in one aspect of the kidney, as determined by regeneration of Bowman's capsules of tubules near the implantation site. However, these changes were mild and did not spread throughout the entire kidney. Without being bound by theory, a hypothesis was proposed that any systemic improvement seen in renal function was due to the action at the individual cell level in rats of group 2 and might not be histologically perceptible enough at the tissue level. Finally, based on the fact that rats treated with the new renal cell prototype No. 1 survived during the test period (3 months), the above study showed that the new renal cell prototype No .1 and the new kidney structure prototype No. 1 extended lifespan. In all groups of nephrectomized rats, weight gain was not possible between the treatment day and the sacrifice day, but in the group treated with the new renal cell prototype No. 1, weight loss was less. .1 and the new kidney structure prototype No. 1 extended lifespan. In all groups of nephrectomized rats, weight gain was not possible between the treatment day and the sacrifice day, but in the group treated with the new renal cell prototype No. 1, weight loss was less.

[0229] Example 8 - Isolation of Renal Cells from a Heterogeneous Cell Population and Enrichment of Specific Bioreactive Renal Cells Isolation of renal cells: Briefly, ten batches of 2-week-old Lewis rat kidneys were obtained from a commercial supplier (Hilltop Lab Animals Inc.) and transported overnight in Viaspan storage medium at a temperature of approximately 4°C. All steps described herein were performed within a biological safety cabinet (BSC) to maintain sterility. The kidneys were washed three times in Hank’s balanced salt solution (HBSS) to rinse out the Viaspan storage medium . After the third wash, the remaining renal capsule and any remaining interstitial tissue were removed. The major calyces were also removed using microdissection techniques. The kidneys were then minced finely using a sterile scalpel and made into a slurry . The slurry was then transferred to 50 mL conical centrifuge tubes and weighed . A small sample was taken for RNA and placed into a sterile 1.5 mL microcentrifuge tube without RNAse and snap frozen in liquid nitrogen. After freezing, it was then transferred to a -80 degree freezer until analysis . The tissue weight of ten young kidneys was equal to approximately 1 gram. Based on the batch weight, the digestion medium was adjusted to deliver 20 mL of digestion medium per gram of tissue . The digestion buffer for this procedure contained 4 units of dispase 1 (STEM Cell Tech), 5 mM CaCl2 (Sigma), and 300 units / mL of type IV collagenase (Worthington) in HBSS . An appropriate volume of pre-warmed digestion buffer was added to the tube, which was then sealed and placed on a rocking machine in an incubator at 37°C for 20 minutes. This first digestion step removed many red blood cells

[0230] ​​​and promote digestion of the remaining tissue. After 20 minutes, the tube was removed and placed in the BSC. The tissue was allowed to settle to the bottom of the tube, and then the supernatant was removed. Next, the remaining tissue was replenished with fresh digestion buffer equal to the starting volume. Again, the tube was placed on a shaker in an incubator at 37°C for an additional 30 minutes. After 30 minutes, the digestion mixture was pipetted through a 70 μm cell strainer (BD Falcon) and placed into an equal volume of neutralization buffer (DMEM w / 10% FBS) to stop the digestion reaction. The cell suspension was then washed by centrifugation at 300 × g for 5 minutes. After centrifugation, the pellet was resuspended in 20 mL of KSFM medium and the sample was obtained for cell counting and viability assessment. The cells were counted and 1 million cells were harvested for RNA, washed in PBS, and snap frozen in liquid nitrogen. The remaining cell suspension was adjusted to 50 mL with KSFM medium and washed again by centrifugation at 300 × g for 5 minutes. After washing, the cell pellet was resuspended at a concentration of 15 million cells per mL of KSFM. Next, 5 milliliters of the kidney cell suspension was added to 5 mL of 30% (w / v) Optiprep® in a 15 mL conical centrifuge tube (BD Falcon) and mixed by inverting 6 times. This formed a final mixture of 15% (w / v) Optiprep®. After inverting, 1 mL of PBS was carefully layered on top of the tube. The tube was centrifuged at 800 × g for 15 minutes without interruption. After centrifugation, the tube was removed and a cell band was formed on top of the mixed gradient. Red blood cells, dead cells, and certain small, less granular cells, in particular

[0231]

[0232] A pellet containing a small population of live cells including cells that produce a fixed EPO, specific tubule cells, and specific endothelial cells. There was also a pellet containing the band. The band was carefully removed using a pipette and transferred to another 1 5 m...

Claims

1. An isolated human kidney cell population for treating kidney diseases, comprising: (i) cells derived from kidney tissue or cultured kidney cells from a kidney sample of a subject, adjusted by fractionating by density gradient separation, wherein the cells are exposed to hypoxic culture conditions prior to fractionation, and (ii) as a result of fractionation, (a) proximal and distal tubule cells, wherein about 90% of the cell population consists of the proximal and distal tubule cells, (b) collecting duct cells, wherein about 10% of the cell population consists of the collecting duct cells, and (c) endocrine cells, vascular cells, and glomerular cells, wherein the endocrine cells, vascular cells, and glomerular cells are present in trace amounts in the cell population, including, an isolated human kidney cell population.

2. The isolated human kidney cell population according to claim 1, wherein the population is adjusted by fractionating cells derived from cultured kidney cells from the kidney sample of the subject.

3. The isolated human kidney cell population according to claim 1 or 2, wherein the population comprises hypoxia-tolerant and iodixanol-tolerant cells.

4. The isolated human kidney cell population according to any one of claims 1 to 3, wherein the population comprises cells capable of receptor-mediated albumin transport.

5. The isolated human kidney cell population according to any one of claims 1 to 4, wherein the population comprises cells expressing hyaluronan synthase 2.

6. The isolated human kidney cell population according to any one of claims 1 to 5, wherein the population comprises cells characterized by the expression of one or more of megalin, cubilin, N-cadherin, and E-cadherin.

7. The isolated human kidney cell population according to any one of claims 1 to 6, wherein the kidney sample is an autologous kidney sample.

8. The isolated human kidney cell population according to any one of claims 1 to 6, wherein the kidney sample is a non-autologous kidney sample.

9. A composition for treating kidney diseases, comprising the isolated human kidney cell population according to any one of claims 1 to 8.

10. A pharmaceutical composition comprising the isolated human kidney cell population according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or excipient.

11. A construct for transplantation into a subject in need of improving kidney function, comprising: a) a biomaterial comprising one or more biocompatible polymers, proteins, or peptides, and b) the isolated human kidney cell population according to any one of claims 1 to 8. The isolated human kidney cell population here is coated on the biological material, deposited on or in the biological material, captured in the biological material, suspended in the biological material, embedded in the biological material, and / or otherwise combined with the biological material, a structure.

12. The biological material is (i) a three-dimensional (3-D) porous biological material suitable for capturing and / or adhering the human kidney cell population, or (ii) a liquid or semi-fluid gel suitable for embedding, adhering, suspending, or coating the human kidney cell population The structure according to claim 11, which is configured as any one of them.

13. The biological material is (i) hyaluronic acid (HA) in the form of a hydrogel, or (ii) hyaluronic acid in the form of a porous foam, or a polylactic acid-based foam having an open cell structure and pores of 50 microns to 300 microns The structure according to claim 11, which is composed of any one of them.

14. The structure according to any one of claims 11 to 13 for treating kidney diseases, anemia, or erythropoietin (EPO) deficiency, providing erythrocyte homeostasis, or improving renal function.

15. A composition for providing erythrocyte homeostasis or improving renal function, comprising the isolated human kidney cell population according to any one of claims 1 to 8.

16. The composition according to claim 9, wherein the kidney disease is accompanied by erythropoietin (EPO) deficiency, and the EPO deficiency is optionally anemia.

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